Biomarker for predicting prognosis of extrahepatic cholangiocarcinoma and application of biomarker

Abstract

The invention discloses a biomarker for predicting prognosis of extrahepatic cholangiocarcinoma and application of the biomarker, and relates to the technical field of biological medicines. The biological marker is a C-X-C motif chemotactic factor ligand 11. According to the invention, an enzyme-linked immunosorbent assay method is adopted to detect the concentration of CXCL11 in bile of a patient with extrahepatic cholangiocarcinoma, so that the prognosis condition of the patient is predicted. A research result shows that the higher the concentration of CXCL11 in bile is, the lower the total survival rate of a patient in one year and three years is, which indicates that the prognosis of the extrahepatic cholangiocarcinoma is poorer. Therefore, it is found that CXCL11 in bile can serve as an effective biomarker and is used for predicting prognosis of extrahepatic cholangiocarcinoma. The discovery provides a brand new biomarker for evaluating the prognosis of the cholangiocarcinoma, and has important clinical significance and scientific research value for accurately predicting the prognosis risk of a patient and expanding the cholangiocarcinoma treatment strategy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a biomarker for predicting the prognosis of extrahepatic bile duct cancer and an application thereof. Background Art

[0002] Cholangiocarcinoma (CCA) is a malignant tumor with high morbidity and mortality. According to the different anatomical sites, it can be divided into intrahepatic cholangiocarcinoma (iCCA), hilar cholangiocarcinoma (pCCA) and distal cholangiocarcinoma (dCCA). Among them, hilar cholangiocarcinoma and distal cholangiocarcinoma are collectively referred to as extrahepatic cholangiocarcinoma. This type of tumor is prone to cause bile duct obstruction and bile stasis, making jaundice its typical clinical manifestation. In recent years, the incidence of cholangiocarcinoma has been on the rise, and there are significant differences between different regions.

[0003] Since the early symptoms of cholangiocarcinoma are atypical, most patients are already in the advanced stage when diagnosed, making accurate prognostic assessment particularly critical. However, existing prognostic assessment methods have certain limitations, such as high invasiveness of the operation or insufficient prediction accuracy. Therefore, it is urgent to develop a more accurate, safe and applicable prognostic prediction method to improve the ability to assess the patient's prognostic risk and further optimize the treatment strategy for cholangiocarcinoma.

[0004] CXCL11 (CXC MotifChemokine Ligand 11) is a chemokine induced by interferon (IFN-γ and IFN-β). It mainly binds to CXCR3 receptors (CXCR3-A and CXCR3-B subtypes) to mediate the chemotaxis of T cells, NK cells and dendritic cells (DC), and plays an important role in immune response and inflammatory response. At present, the role of CXCL11 in extrahepatic cholangiocarcinoma is still unclear, and there is still a lack of relevant research reports on the relationship between its expression level and patient prognosis, which urgently needs further exploration. Summary of the invention

[0005] The purpose of the present invention is to provide a biomarker for predicting the prognosis of extrahepatic bile duct cancer and its application to solve the problems existing in the above-mentioned prior art. The present invention has found that CXC motif chemokine ligand 11 in bile can be used as a biomarker for predicting the prognosis of extrahepatic bile duct cancer, providing a new marker for evaluating the prognosis of bile duct cancer, which is of great significance for accurately predicting the prognosis risk of patients and enriching the treatment strategy of bile duct cancer.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma. The biomarker is CXC motif chemokine ligand 11.

[0008] Furthermore, an elevated concentration of the biomarker in the bile of patients with extrahepatic cholangiocarcinoma indicates a poor prognosis for these patients.

[0009] The present invention also provides the use of a reagent for detecting the concentration of C-X-C motif chemokine ligand 11 in bile in the preparation of a product for predicting the prognosis of extrahepatic cholangiocarcinoma.

[0010] Furthermore, the product includes a reagent or a kit.

[0011] The present invention also provides a product for predicting the prognosis of extrahepatic cholangiocarcinoma, comprising a reagent for detecting the concentration of C-X-C motif chemokine ligand 11 in bile.

[0012] Furthermore, the product includes a reagent or a kit.

[0013] Furthermore, the product detects the concentration of C-X-C motif chemokine ligand 11 in bile by enzyme-linked immunosorbent assay.

[0014] The present invention discloses the following technical effects:

[0015] The present invention uses the enzyme-linked immunosorbent assay method to detect the concentration of CXCL11 in the bile of patients with extrahepatic cholangiocarcinoma, so as to predict the prognosis of the patients. The research results show that the higher the concentration of CXCL11 in the bile, the lower the overall survival rate of the patients at 1 year and 3 years, which indicates a poor prognosis for extrahepatic cholangiocarcinoma. Therefore, the present invention discovers that CXCL11 in bile can be used as an effective biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma. This discovery provides a new biomarker for evaluating the prognosis of cholangiocarcinoma and has important clinical significance and scientific research value for accurately predicting the prognosis risk of patients and expanding the treatment strategies for cholangiocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figure 1 Shows the concentration difference of CXCL11 in the bile of 129 patients with extrahepatic cholangiocarcinoma and 60 patients with cholelithiasis in Example 1;

[0018] Figure 2 Shows the receiver operating characteristic curve of patients with extrahepatic cholangiocarcinoma drawn using the CXCL11 concentration and survival status in Example 2;

[0019] Figure 3 It is a survival curve graph of the overall survival (OS) for discriminating the CXCL11 concentration in the samples of patients with extrahepatic cholangiocarcinoma in Example 2. Detailed implementation manners

[0020] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0023] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0024] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0025] Example 1 Determination of the CXCL11 concentration in bile for predicting the prognosis of extrahepatic cholangiocarcinoma

[0026] 1. Source of bile from human extrahepatic cholangiocarcinoma

[0027] Bile (129 cases) from patients diagnosed with extrahepatic cholangiocarcinoma in Qilu Hospital of Shandong University from January 2018 to December 2023 was collected. All these patients underwent radical resection of cholangiocarcinoma and had a smooth postoperative recovery without related complications. Additionally, bile from patients with cholelithiasis (60 cases) was collected as a control group.

[0028] Inclusion criteria:

[0029] (1) Preoperative imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examinations confirmed extrahepatic cholangiocarcinoma or cholelithiasis.

[0030] (2) The patient was in good general condition without severe organic lesions in important organs such as the heart, lungs, kidneys, and brain that would affect the implementation of the operation and postoperative recovery.

[0031] (3) The operation was successful without severe postoperative complications, the postoperative recovery was smooth, and no secondary operation related to the primary disease was performed.

[0032] (4) Clear clinical data, postoperative follow-up data, and postoperative survival time, with a postoperative survival time of more than 3 months.

[0033] Exclusion criteria:

[0034] (1) History of previous upper abdominal surgery.

[0035] (2) Those who underwent palliative surgery or termination of the operation due to accidents such as massive hemorrhage or cardiac arrest during the operation.

[0036] (3) Those who underwent palliative surgery due to local organ infiltration or extensive intra-abdominal organ metastasis found during the operation.

[0037] (4) Those who died due to surgery-related complications or other non-surgical accidents after the operation, or those with a postoperative survival period of less than 3 months.

[0038] 2. Determination of CXCL11 concentration in bile

[0039] The concentration of CXCL11 in the bile of patients with cholangiocarcinoma and cholelithiasis was determined using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0040] (1) Sample preparation: Thaw and dilute the bile sample 10 times.

[0041] (2) Pretreatment of the enzyme-linked immunosorbent assay plate: Use the CXCL11 assay kit (R&D Systems, NO. DCX110) to detect the concentration of CXCL11. Dilute the CXCL11 antibody and add it to the wells of the enzyme-linked immunosorbent assay plate, then incubate overnight at 4°C.

[0042] (3) Washing of the enzyme-linked immunosorbent assay plate: Use the washing solution to wash the excess antibody on the enzyme-linked immunosorbent assay plate to reduce non-specific binding.

[0043] (4) Sample and standard addition: Add appropriate amounts of the sample and standard to different wells of the microplate. The standard is used to establish a concentration curve for the quantitative determination of the CXCL11 concentration. After that, incubate the microplate at room temperature for 2 hours.

[0044] (5) Microplate washing: Similar to step (3), wash the microplate with the washing solution to remove unbound sample and standard.

[0045] (6) Detection antibody addition: Add the detection antibody, i.e., biotinylated secondary antibody, to the wells of the microplate. These antibodies will specifically bind to the target protein. Incubate again at room temperature for 1 hour.

[0046] (7) Microplate washing: Wash the microplate with the washing solution to remove unbound detection antibody.

[0047] (8) Enzyme conjugate addition: Add the enzyme conjugate (usually streptavidin protein A labeled with horseradish peroxidase (HRP)) to the wells of the microplate. The enzyme conjugate will bind to the biotinylated detection antibody. Operate according to the kit instructions, usually incubate at room temperature for 30 minutes to 1 hour.

[0048] (9) Microplate washing: Wash the microplate again with the washing solution to remove unbound enzyme conjugate.

[0049] (10) Chromogenic substrate addition: Add the chromogenic substrate, i.e., TMB, to each well of the microplate. The substrate reacts with the enzyme to produce a color change. Incubate at room temperature for 10 - 30 minutes until the color develops significantly.

[0050] (11) Stop the reaction: After the appropriate color development, add the stop solution (sulfuric acid or phosphoric acid) to terminate the reaction. The color will change from blue to yellow.

[0051] (12) Read the absorbance: Use a microplate reader to read the absorbance value of each well (at a wavelength of 450 nm).

[0052] (13) Data analysis: Use the absorbance values of the standard to establish a standard curve. Then, convert the absorbance value of the sample to the CXCL11 concentration according to this curve.

[0053] The results are as Figure 1 shown. There is a significant difference in the CXCL11 concentration between the bile of extrahepatic cholangiocarcinoma and cholelithiasis. The CXCL11 concentration in the bile of patients with extrahepatic cholangiocarcinoma is significantly higher than that of patients with cholelithiasis.

[0054] Example 2 Predicting the prognosis of extrahepatic cholangiocarcinoma by combining the CXCL11 concentration in bile

[0055] The receiver operating characteristic curve was plotted based on the measured CXCL11 concentration and the patient survival status (P = 0.001, AUC = 0.719), and the results are shown in Figure 2 . The Cut-off value (852.3695 pg / mL) was calculated according to the receiver operating characteristic curve for binary classification, and the cohort was divided into a high CXCL11 concentration group and a low CXCL11 concentration group. That is, when the CXCL11 concentration ≥ 852.3695 pg / mL, it was defined as the high CXCL11 concentration group, and when the CXCL11 concentration < 852.3695 pg / mL, it was defined as the low CXCL11 concentration group.

[0056] As Figure 3 shown, the concentration of CXCL11 in the bile of extrahepatic cholangiocarcinoma was positively correlated with the poor prognosis of patients. That is, the overall survival trend of patients in the high bile CXCL11 concentration group was lower than that of patients in the low CXCL11 concentration group, and there was a significant difference in their 3-year overall survival rate, indicating that this model could effectively predict the prognosis of patients. Univariate analysis was used to reveal the prognostic predictive value of bile CXCL11 concentration, and the results are shown in Table 1.

[0057] Table 1 Prognostic significance of clinicopathological characteristics of patients with extrahepatic cholangiocarcinoma

[0058]

[0059] Note: P: log-rank test.

[0060] Based on the above research and analysis results, the following method for prognostic assessment of extrahepatic cholangiocarcinoma can be obtained:

[0061] S1: Extract the bile of patients with extrahepatic cholangiocarcinoma after surgery and perform pre-treatment of the sample experiment;

[0062] S2: Detect the concentration of CXCL11 in the bile by ELISA method;

[0063] S3: Predict the prognosis of patients with extrahepatic cholangiocarcinoma according to the detection result of the CXCL11 concentration: high concentration of CXCL11 in the bile (≥ 852.3695 pg / mL) indicates poor prognosis of the patient; low concentration of CXCL11 in the bile (< 852.3695 pg / mL) indicates good prognosis of the patient.

[0064] Based on the above experimental results, it was found that the present invention could directly and effectively predict the prognosis of patients with extrahepatic cholangiocarcinoma according to the CXCL11 concentration in the bile of patients. The higher the concentration, the worse the prognosis of the patient.

[0065] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma, characterized in that, The biomarker is C-X-C motif chemokine ligand 11.

2. The biomarker according to claim 1, wherein An increase in the concentration of the biomarker in the bile of patients with extrahepatic cholangiocarcinoma indicates a poor prognosis for the patients with extrahepatic cholangiocarcinoma.

3. Use of a reagent for detecting the concentration of C-X-C motif chemokine ligand 11 in bile in the preparation of a product for predicting the prognosis of extrahepatic cholangiocarcinoma.

4. The application according to claim 3, characterized in that, The product includes a reagent or a kit.

5. A product for predicting the prognosis of extrahepatic cholangiocarcinoma, characterized in that, It includes a reagent for detecting the concentration of C-X-C motif chemokine ligand 11 in bile.

6. The product according to claim 5, characterized in that, The product includes a reagent or a kit.

7. The product according to claim 5, characterized in that, The product detects the concentration of the C-X-C motif chemokine ligand 11 in bile by enzyme-linked immunosorbent assay.

Images