A biomarker for predicting prognosis of extrahepatic cholangiocarcinoma and application thereof

Abstract

The application discloses a biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma and application thereof, and relates to the technical field of biological medicine.The biomarker is C-X-C motif chemokine ligand 11.The application adopts an enzyme-linked immunosorbent assay method to detect the concentration of CXCL11 in bile of an extrahepatic cholangiocarcinoma patient, so as to predict the prognosis of the patient.The research result shows that the higher the concentration of CXCL11 in the bile is, the lower the overall survival rate of the patient in 1 year and 3 years is, which indicates that the prognosis of the extrahepatic cholangiocarcinoma is poor.Therefore, the application finds that the CXCL11 in the bile can be used as an effective biomarker for predicting the prognosis of the extrahepatic cholangiocarcinoma.This finding provides a brand-new biomarker for evaluating the prognosis of cholangiocarcinoma, and has important clinical significance and scientific research value for accurately predicting the prognosis risk of the patient and expanding the treatment strategy of the cholangiocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma and its application. Background Technology

[0002] Cholangiocarcinoma (CCA) is a malignant tumor with high incidence and mortality rates. Based on anatomical location, it can be divided into intrahepatic cholangiocarcinoma (iCCA), hilar cholangiocarcinoma (pCCA), and distal cholangiocarcinoma (dCCA). Hilar cholangiocarcinoma and distal cholangiocarcinoma are collectively referred to as extrahepatic cholangiocarcinoma. This type of tumor easily causes biliary obstruction and cholestasis, making jaundice a typical clinical manifestation. In recent years, the incidence of cholangiocarcinoma has been on the rise, and there are significant differences between different regions.

[0003] Because early symptoms of cholangiocarcinoma are often atypical, most patients are diagnosed at an advanced stage, making accurate prognostic assessment crucial. However, existing prognostic assessment methods have limitations, such as being highly invasive or lacking sufficient predictive accuracy. Therefore, there is an urgent need to develop a more accurate, safe, and widely applicable prognostic prediction method to improve the ability to assess patient prognostic risks and further optimize treatment strategies for cholangiocarcinoma.

[0004] CXCL11 (CXC Motif Chemokine Ligand 11) is a chemokine induced by interferon (IFN-γ and IFN-β). It mediates chemotaxis of T cells, NK cells, and dendritic cells (DCs) primarily through binding to CXCR3 receptors (CXCR3-A and CXCR3-B isoforms), playing a crucial role in immune responses and inflammatory reactions. Currently, the role of CXCL11 in extrahepatic cholangiocarcinoma remains unclear, and the relationship between its expression level and patient prognosis lacks sufficient research reports, requiring further investigation. Summary of the Invention

[0005] The purpose of this invention is to provide a biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma and its application, thereby addressing the problems existing in the prior art. This invention discovers that CXC motif chemokine ligand 11 in bile can serve as a biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma, providing a novel biomarker for assessing the prognosis of cholangiocarcinoma. This is of great significance for accurately predicting the prognostic risk of patients and enriching treatment strategies for cholangiocarcinoma.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma, wherein 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 those patients.

[0009] The present invention also provides the application of a reagent for detecting the concentration of CXC motif chemokine ligand 11 in bile in the preparation of products for predicting the prognosis of extrahepatic cholangiocarcinoma.

[0010] Furthermore, the product includes reagents or kits.

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

[0012] Furthermore, the product includes reagents or kits.

[0013] Furthermore, the concentration of the CXC motif chemokine ligand 11 in bile was detected by enzyme-linked immunosorbent assay (ELISA).

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

[0015] This invention employs an enzyme-linked immunosorbent assay (ELISA) to detect the concentration of CXCL11 in the bile of patients with extrahepatic cholangiocarcinoma, thereby predicting patient prognosis. The results showed that higher CXCL11 concentrations in bile were associated with lower 1-year and 3-year overall survival rates, indicating a poorer prognosis for extrahepatic cholangiocarcinoma. Therefore, this invention discovers that CXCL11 in bile can serve as an effective biomarker for predicting the prognosis of extrahepatic cholangiocarcinoma. This finding provides a novel biomarker for assessing the prognosis of cholangiocarcinoma and has significant clinical and research value for accurately predicting patient prognostic risk and expanding treatment strategies for cholangiocarcinoma. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0018] Figure 2 The receiver operating characteristic curves (ROCs) of extrahepatic cholangiocarcinoma patients were plotted using CXCL11 concentration and survival status in Example 2.

[0019] Figure 3 This is a survival curve of overall survival (OS) in extrahepatic bile duct carcinoma patients with CXCL11 concentration in Example 2. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Measurement of bile CXCL11 concentration for predicting the prognosis of extrahepatic cholangiocarcinoma

[0026] 1. Source of bile in human extrahepatic bile duct carcinoma

[0027] Bile samples were collected from 129 patients diagnosed with extrahepatic cholangiocarcinoma at Qilu Hospital of Shandong University between January 2018 and December 2023. All of these patients underwent radical resection of cholangiocarcinoma and recovered smoothly postoperatively without any related complications. Bile samples were also collected from 60 patients with cholelithiasis as a control group.

[0028] Inclusion criteria:

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

[0030] (2) The patient is in good general condition and has no serious organic lesions in important organs such as heart, lungs, kidneys, and brain that would affect the implementation of the surgery and postoperative recovery.

[0031] (3) The surgery was successful, with no serious postoperative complications, and the postoperative recovery was smooth. No secondary surgery 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 upper abdominal surgery.

[0035] (2) Patients who undergo palliative surgery or have their surgery terminated due to unexpected events such as massive bleeding or cardiac arrest during the operation.

[0036] (3) Patients who undergo palliative surgery due to the discovery of local organ infiltration or extensive organ metastasis in the abdominal cavity during the operation.

[0037] (4) Those who die after surgery due to surgical complications or other non-surgical accidents, or whose postoperative survival period is 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 an enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

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

[0041] (2) Pretreatment of microplates: The concentration of CXCL11 was detected using a CXCL11 assay kit (R&D Systems, NO. DCX110). The CXCL11 antibody was diluted and added to the wells of the microplate, and then incubated overnight at 4°C.

[0042] (3) Washing the ELISA plate: Use washing solution to wash away excess antibodies on the ELISA plate to reduce non-specific binding.

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

[0044] (5) Washing the ELISA plate: Similar to step (3), wash the ELISA plate with washing solution to remove unbound samples and standards.

[0045] (6) Addition of detection antibodies: Add the detection antibodies, i.e., biotin-labeled secondary antibodies, to the wells of the ELISA plate. These antibodies will specifically bind to the target protein. Incubate again at room temperature for 1 hour.

[0046] (7) Washing the ELISA plate: Wash the ELISA plate with washing solution to remove unbound detection antibodies.

[0047] (8) Enzyme-linked addition: Add the enzyme conjugate (usually horseradish peroxidase (HRP)-labeled streptomycin A) to the wells of the ELISA plate. The enzyme conjugate will bind to the biotin-labeled detection antibody. Follow the kit instructions, and incubate at room temperature for 30 minutes to 1 hour.

[0048] (9) Washing the ELISA plate: Wash the ELISA plate again with washing solution to remove unbound enzymes.

[0049] (10) Addition of chromogenic substrate: Add the chromogenic substrate, TMB, to each well of the microplate. The substrate reacts with the enzyme, producing 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 is achieved, add a stopping solution (sulfuric acid or phosphoric acid) to terminate the reaction. The color will change from blue to yellow.

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

[0052] (13) Data analysis: A standard curve was established using the absorbance values ​​of the standards. Then, the absorbance values ​​of the samples were converted into the concentration of CXCL11 based on this curve.

[0053] The results are as follows Figure 1 As shown, there was a significant difference in the concentration of CXCL11 in the bile of patients with extrahepatic cholangiocarcinoma and patients with cholelithiasis. The concentration of CXCL11 in the bile of patients with extrahepatic cholangiocarcinoma was significantly higher than that of patients with cholelithiasis.

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

[0055] Receiver operating characteristic (ROC) curves were plotted based on the measured CXCL11 concentration and patient survival status (P = 0.001, AUC = 0.719). The results are shown below. Figure 2 The cut-off value (852.3695 pg / mL) was calculated based on the receiver operating characteristic curve and then divided into two groups: a high-concentration CXCL11 group and a low-concentration CXCL11 group. Specifically, a CXCL11 concentration ≥ 852.3695 pg / mL was defined as the high-concentration CXCL11 group, and a CXCL11 concentration < 852.3695 pg / mL was defined as the low-concentration CXCL11 group.

[0056] like Figure 3 As shown, the concentration of CXCL11 in bile from extrahepatic cholangiocarcinoma patients is positively correlated with poor prognosis. Specifically, patients with high CXCL11 concentrations in bile have a lower overall survival rate than those with low CXCL11 concentrations, with a significant difference in 3-year overall survival. This indicates that the model can effectively predict patient prognosis. Univariate analysis was used to reveal the prognostic predictive value of bile CXCL11 concentration; the results are shown in Table 1.

[0057] Table 1. Prognostic significance of clinicopathological features in patients with extrahepatic bile duct carcinoma.

[0058]

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

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

[0061] S1: Extract bile from patients with extrahepatic bile duct cancer after surgery and perform sample pretreatment for experiments;

[0062] S2: The concentration of CXCL11 in bile was detected using the ELISA method;

[0063] S3: Predict the prognosis of patients with extrahepatic cholangiocarcinoma based on the concentration of CXCL11: High concentration of CXCL11 in bile (≥852.3695 pg / mL) indicates a poor prognosis; low concentration of CXCL11 in bile (<852.3695 pg / mL) indicates a better prognosis.

[0064] Based on the above experimental results, it was found that the present invention can directly and effectively predict the prognosis of patients with extrahepatic cholangiocarcinoma by measuring the concentration of CXCL11 in their bile; the higher the concentration, the worse the prognosis.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of reagents for detecting the concentration of CXC motif chemokine ligand 11 in bile in the preparation of products for predicting the prognosis of extrahepatic cholangiocarcinoma.

2. The application according to claim 1, characterized in that, The products include reagents or kits.

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