A molecular marker BST2 for predicting the prognosis of cholangiocarcinoma immunotherapy and its application

Abstract

The present invention discloses a molecular marker BST2 for predicting the prognosis of immunotherapy for bile duct cancer and its application, belonging to the field of biomedical technology. The present invention can make a judgment on the early diagnosis of bile duct cancer patients by detecting the expression level of BST2 in extracellular microvesicles separated from the bile of bile duct cancer patients. BST2 is highly expressed in extracellular microvesicles separated from the bile of bile duct cancer patients, while it is lowly expressed in extracellular microvesicles separated from the bile of cholelithiasis patients. Therefore, the expression level of the BST2 molecular marker in extracellular microvesicles separated from the bile of bile duct cancer patients can be detected to predict the degree of neural invasion of bile duct cancer patients. The treatment plan can be selected and modified based on the prediction, thereby improving the patient's survival time and quality of life, and having great economic value and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a molecular marker BST2 for predicting the prognosis of cholangiocarcinoma immunotherapy and an application thereof. Background Art

[0002] Cholangiocarcinoma is a malignant tumor that originates from bile duct epithelial cells. Treatment options include surgical resection, radiotherapy, and chemotherapy. However, because early symptoms of cholangiocarcinoma are not obvious, many patients are diagnosed in the late stages, making surgical resection less likely.

[0003] In recent decades, the overall prognosis of bile duct cancer has almost stagnated. Radical surgical resection is one of the most effective methods for treating early-stage bile duct cancer. However, the early clinical symptoms of bile duct cancer are not obvious, and it is usually diagnosed only when bile duct obstruction is followed by symptoms such as jaundice and abdominal pain. As a result, about 65% of patients lose the opportunity for radical surgery, and their one-year survival rate is only 10%-20%. In terms of chemotherapy, the NCCN guidelines recommend systemic chemotherapy based on gemcitabine combined with cisplatin, but the five-year survival rate of patients with advanced bile duct cancer using this chemotherapy regimen is less than 5%. For advanced bile duct cancer, there is currently no standardized and recognized chemotherapy regimen that can prolong survival time. Various factors affect the prognosis of bile duct cancer patients. Therefore, finding new markers that can predict the prognosis of bile duct cancer is of great significance for accurately predicting patients' prognostic risks and enriching bile duct cancer treatment strategies.

[0004] Extracellular vesicles (EVs) comprise a heterogeneous population of membrane vesicles of various origins. These EVs are named based on their origin, size, morphology, and cargo content and can be divided into two distinct categories: exosomes and microvesicles. EVs are formed by budding from the plasma membrane, in which case they are termed microvesicles, or by intraluminal vesicles (ILVs) within the lumen of multivesicular endosomes (MVEs). MVEs fuse with the plasma membrane to release ILVs, which are then termed exosomes. Studies of EV composition have revealed that they can carry a variety of cargoes, including proteins, lipids, and nucleic acids, and that this cargo can vary significantly between cells and conditions, with the specific composition directly influencing the fate and function of EVs. Notably, depending on the cell type, EVs display a panel of cell-type-specific proteins that explain their specific fate and function. Despite their distinct modes of biogenesis, exosomes and microvesicles display similar appearance, overlapping sizes, and generally shared composition, making their origin difficult to determine once isolated from the extracellular medium or biological fluids. Among the studies on extracellular vesicles, exosomes have been studied more, however, the mechanism by which microvesicles play a role in cancer remains to be explored.

[0005] Bone marrow stromal cell antigen 2 (BST2), also known as CD317 or HM1.24, is located on chromosome 19p13.11 and is a well-known cellular restriction factor that prevents the release of HIV-1, Kaposi's sarcoma-associated herpesvirus, Ebola virus, and other enveloped viruses from the cell surface. BST2 was initially identified as a type II transmembrane glycoprotein expressed in bone marrow stromal cell lines and reportedly involved in the growth of pre-B cells and in the innate immune response. Multiple studies have demonstrated aberrant expression of BST2 in various cancers, including breast, oral, lung, chronic lymphocytic leukemia, and gastrointestinal cancer cells. For example, BST2 functions as an anti-apoptotic factor in solid tumors through the mitochondrial apoptosis-inducing factor axis, promoting the migration, invasion, and invadopodia formation of breast cancer cells. The transcription factor SP1 activates BST2 to accelerate the proliferation and migration of pancreatic cancer cells. However, how BST2 regulates the progression of cholangiocarcinoma (CCA) remains largely unexplored. Summary of the Invention

[0006] The present invention aims to provide a molecular marker, BST2, for predicting the prognosis of cholangiocarcinoma immunotherapy and its application, to address the aforementioned problems of the prior art. The present invention screens for differentially expressed protein markers by mass spectrometry analysis of bile-derived microvesicles. By measuring the expression levels of specific protein markers and comparing them with reference levels, the prognosis of cholangiocarcinoma patients can be predicted. Based on these research findings, the present invention was completed.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a molecular marker for predicting the prognosis of cholangiocarcinoma immunotherapy, wherein the molecular marker is bone marrow stromal cell antigen 2.

[0009] The second technical solution of the present invention is the use of the molecular marker as a detection target in the preparation of a product for predicting and / or diagnosing bile duct cancer.

[0010] The third technical solution of the present invention is the use of a substance for detecting the expression level of the molecular marker bone marrow stromal cell antigen 2 in peripheral blood serum microvesicles in the preparation of a product.

[0011] A fourth technical solution of the present invention is a product for predicting and / or diagnosing bile duct cancer, wherein the product uses the molecular marker as a detection target.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] This study discovered that BST2 can be used as a molecular marker for predicting the prognosis of cholangiocarcinoma. Patients with high BST2 expression in bile microvesicles from cholangiocarcinoma had a significantly lower overall survival rate than those with low BST2 expression. This study provides a new marker for predicting the prognosis of cholangiocarcinoma and formulating treatment plans, which is of great significance for accurately predicting patients' prognostic risks and enriching cholangiocarcinoma treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a survival analysis of the different expressions of BST2 in bile microvesicles of cholangiocarcinoma patients in Example 2 of the present invention.

[0016] Figure 2 is the relative expression level of BST2 protein in bile microvesicles of patients with cholelithiasis and cholangiocarcinoma in mass spectrometry in Example 1 of the present invention.

[0017] Figure 3 The expression of BST2 in bile microvesicles of the control group (cholelithiasis) and cholangiocarcinoma patients in Example 2 of the present invention.

[0018] Figure 4 Figure 3 is the ROC curve of BST2 expression level and neuroinvasion. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0025] An embodiment of the present invention provides a molecular marker for predicting the prognosis of cholangiocarcinoma immunotherapy, wherein the molecular marker is bone marrow stromal cell antigen 2.

[0026] The biomarker can be used to determine the prognosis of patients with bile duct cancer. The present invention found that BST2 is abnormally highly expressed in microvesicles secreted in the bile of patients with bile duct cancer, and the expression level of BST2 in the microvesicles is closely correlated with the overall survival of the patients. Specifically, the expression level of BST2 in the microvesicles secreted in the bile is negatively correlated with the overall survival of patients with bile duct cancer.

[0027] The embodiments of the present invention also provide the use of the molecular marker as a detection target in the preparation of a product for predicting and / or diagnosing bile duct cancer.

[0028] The embodiments of the present invention also provide the use of BST2 as a target in the preparation of products for tumor treatment, tumor prognosis, and prediction of tumor treatment effects.

[0029] The embodiments of the present invention also provide the use of a substance for detecting the expression level of the molecular marker bone marrow stromal cell antigen 2 in peripheral blood serum microvesicles in the preparation of a product.

[0030] In some specific embodiments, the substance includes but is not limited to substances for detecting the expression level of the molecular marker BST2 in bile microvesicles by mass spectrometry or ELISA enzyme-linked immunosorbent assay.

[0031] An embodiment of the present invention further provides a product for predicting and / or diagnosing bile duct cancer, wherein the product uses the molecular marker as a detection target.

[0032] In some specific embodiments, the product comprises a reagent or a kit.

[0033] In some specific embodiments, the kit contains a reagent for detecting BST2.

[0034] In some specific embodiments, the reagent for detecting BST2 includes at least one of a reagent for detecting whether the BST2 gene is overexpressed, a reagent for quantitatively detecting the RNA transcription level of BST2, and a reagent for quantitatively detecting the protein expression level of BST2.

[0035] In some specific embodiments, the product uses a blood specimen of a patient to be tested as the test sample.

[0036] In some specific embodiments, the function of the product is to evaluate the prognosis of patients with bile duct cancer, and the prognosis is the overall survival of the patient.

[0037] An embodiment of the present invention further provides a device, comprising: one or more devices for detecting the above-mentioned biomarkers.

[0038] An embodiment of the present invention further provides a device for evaluating the prognosis of bile duct cancer, comprising:

[0039] i) an analysis module, the analysis module comprising: a detection agent for determining the expression level of a biomarker selected from the above in a sample of a subject, and;

[0040] ii) an evaluation module, comprising determining the prognosis of the subject according to the expression levels of the biomarkers determined in i).

[0041] The present invention can make judgments for the early diagnosis of bile duct cancer patients by detecting the expression level of BST2 in extracellular microvesicles isolated from the bile of bile duct cancer patients. BST2 is highly expressed in extracellular microvesicles isolated from the bile of bile duct cancer patients, while it is lowly expressed in extracellular microvesicles isolated from the bile of cholelithiasis patients. Therefore, detecting the expression level of the BST2 molecular marker in extracellular microvesicles isolated from the bile of bile duct cancer patients can predict the degree of neuroinvasive tumor in bile duct cancer patients. Based on the prediction, treatment plans can be selected and modified, thereby improving the survival time and quality of life of patients, with great economic value and social benefits.

[0042] Example 1

[0043] 1. Screening of protein molecules

[0044] 1.1 Sample Source

[0045] Inclusion criteria:

[0046] (1) Male or female patients aged 18-80 years.

[0047] (2) Cholangiocarcinoma is confirmed by puncture combined with imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examination.

[0048] (3) ECOG score 0-1, Child-Pugh score A or B.

[0049] (4) Clear clinical data, follow-up data and survival time, with the survival time after treatment being greater than 3 months.

[0050] Exclusion criteria:

[0051] (1) Hematological examination: WBC < 4.0*10 9 / L,HB<80g / L,PLT<75*10 9 / L.

[0052] (2) Coagulation function: international normalized (prothrombin time) ratio (INR) > 2.3, or prothrombin time (PT) prolonged > 6 seconds.

[0053] (3) Liver function indicators: bile albumin (ALB) <2.8 g / dl, total bile bilirubin (TBIL) >51.3 μmo1 / , alkaline phosphatase, bile transaminase (ALT and AST) >5 times the upper limit of normal value.

[0054] (4) Renal function index: Bile creatinine (CRE) > 1.5 times the upper limit of normal value

[0055] (5) The subject has experienced acute cardiovascular and cerebrovascular diseases such as acute cerebral infarction, acute coronary syndrome, etc. within one month, and the cardiovascular clinical symptoms or diseases cannot be well controlled.

[0056] (6) Ascites, hepatic encephalopathy, Gilbert's syndrome, sclerosing cholangitis, etc.

[0057] Bile specimens from 10 patients with cholangiocarcinoma and 10 patients without cholangiocarcinoma were collected, and microvesicles and exosomes in the bile were separated by ultraspeed gradient centrifugation.

[0058] 1.2 Mass spectrometry

[0059] Mass spectrometry is used to detect all protein molecules in the bile extracellular vesicles of step (1), identify existing and newly discovered protein molecules, and screen protein molecules that are significantly differently expressed in the bile of cholangiocarcinoma patients compared with the bile of non-cholangiocarcinoma patients.

[0060] The results showed that the expression of BST2 in the cholangiocarcinoma group was significantly higher than that in the non-cholangiocarcinoma group. Figure 2 .

[0061] Example 2

[0062] 1. Detection of the relative expression of BST2 in bile extracellular vesicles of patients with cholangiocarcinoma

[0063] 1.1 Sample Source

[0064] Inclusion criteria:

[0065] (1) Male or female patients aged 18-80 years.

[0066] (2) Cholangiocarcinoma is confirmed by puncture combined with imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examination.

[0067] (3) ECOG score 0-1, Child-Pugh score A or B.

[0068] (4) Clear clinical data, follow-up data and survival time, with the survival time after treatment being greater than 3 months.

[0069] Exclusion criteria:

[0070] (1) Hematological examination: WBC < 4.0*10 9 / L,HB<80g / L,PLT<75*10 9 / L.

[0071] (2) Coagulation function: international normalized (prothrombin time) ratio (INR) > 2.3, or prothrombin time (PT) prolonged > 6 seconds.

[0072] (3) Liver function indicators: bile albumin (ALB) <2.8 g / dl, total bile bilirubin (TBIL) >51.3 μmo1 / , alkaline phosphatase, bile transaminase (ALT and AST) >5 times the upper limit of normal value.

[0073] (4) Renal function indicators: bile creatinine (CRE)>1.5 times the upper limit of normal value.

[0074] (5) The subject has experienced acute cardiovascular and cerebrovascular diseases such as acute cerebral infarction, acute coronary syndrome, etc. within one month, and the cardiovascular clinical symptoms or diseases cannot be well controlled.

[0075] (6) Ascites, hepatic encephalopathy, Gilbert's syndrome, sclerosing cholangitis, etc.

[0076] Bile specimens from 35 patients with cholangiocarcinoma and 35 patients without cholangiocarcinoma were collected clinically. The relative content of BST2 was detected by ELISA and the differences were analyzed.

[0077] 1.2 Determination of BST2 expression

[0078] (1) Extracellular vesicles in bile were separated by ultracentrifugation, and total proteins of exosomes and microvesicles were extracted.

[0079] (2) ELISA was used to detect the BST2 content in the protein extracted in step (1). The results are shown in Figure 3 .

[0080] 2. Survival Analysis

[0081] All samples were divided into high-expression and low-expression BST2 groups, and the overall survival of all patients was followed up to evaluate the impact of the expression level of this protein on patient prognosis.

[0082] 3. Evaluation of the relationship between BST2 expression level and neuroinvasion is shown in Table 1.

[0083] Table 1

[0084] BST2 low expression group (n=15) BST2 high expression group (n=20) Neuroinvasive 5 17 No neuroinvasive 10 3

[0085] Chi-square test: P=0.007.

[0086] ROC curve and area under the curve are shown in Figure 4 Using bile microvesicles (BST2) to screen for cholangiocarcinoma, the area under the receiver operating characteristic (ROC) curve was 0.776. At a cutoff value of 429.7427, the sensitivity and specificity were 77.3% and 76.9%, respectively. A value greater than 429.7427 indicates a high likelihood of perineural invasion in patients with cholangiocarcinoma, prompting further pathological diagnosis.

[0087] The results showed that the overall survival rate of the BST2 high expression group was significantly lower than that of the BST2 low expression group. There were also significant differences in the 1-year and 3-year overall survival (OS). Figure 1 These results indicate that bile vesicle BST2 can be used as a molecular marker to predict the prognosis of cholangiocarcinoma.

[0088] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of bone marrow stromal cell antigen 2 in bile microvesicles as a detection target in the preparation of products for prognosis and / or diagnosis of cholangiocarcinoma.

2. Use of a substance for detecting the expression level of the molecular marker bone marrow stromal cell antigen 2 in bile microvesicles in the preparation of a product for prognosis and / or diagnosis of bile duct cancer.

Images