Molecular marker BST2 for predicting immunotherapy prognosis of bile duct cancer and application of molecular marker BST2

Abstract

The invention discloses a molecular marker BST2 for predicting immunotherapy prognosis of bile duct cancer and application of the molecular marker BST2, and belongs to the technical field of biomedicine. By detecting the expression level of the BST2 in the extracellular microvesicles separated from the bile of the bile duct cancer patient, early diagnosis of the bile duct cancer patient can be judged, the BST2 is highly expressed in the extracellular microvesicles separated from the bile of the bile duct cancer patient, and the BST2 is low in expression in the extracellular microvesicles separated from the bile of the cholelithiasis patient. Therefore, by detecting the expression quantity of the BST2 molecular marker in the extracellular microvesicles separated from the bile of the bile duct cancer patient to predict the nerve invasion tumor degree of the bile duct cancer patient, a treatment scheme can be selected and changed according to prediction, the survival time and the survival quality of the patient are improved, and the method has relatively high economic value and social benefit.

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 originating from bile duct epithelial cells. Its treatment methods mainly include surgical resection, radiotherapy, chemotherapy, etc. However, since the early symptoms of cholangiocarcinoma are not obvious, many patients are diagnosed in the late stage, and the possibility of surgical resection is reduced.

[0003] In recent decades, the overall prognosis of cholangiocarcinoma has almost stagnated. Radical surgical resection is one of the most effective methods for treating early cholangiocarcinoma. However, the early clinical symptoms of cholangiocarcinoma are not obvious, and it is usually diagnosed only when the bile duct is obstructed and then jaundice, abdominal pain and other symptoms appear, which makes 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 cholangiocarcinoma using this chemotherapy regimen is less than 5%. For advanced cholangiocarcinoma, there is currently no standardized and recognized chemotherapy regimen that can prolong survival time. Various factors affect the prognosis of patients with cholangiocarcinoma. Therefore, finding new markers that can predict the prognosis of cholangiocarcinoma is of great significance for accurately predicting the prognostic risk of patients and enriching the treatment strategy of cholangiocarcinoma.

[0004] Extracellular vesicles include a heterogeneous population of membrane vesicles of various origins. Extracellular vesicles are named according to their origin, size, morphology and content, and can now be divided into two different categories: exosomes and microvesicles. Extracellular vesicles are formed by budding from the plasma membrane, in which case they are called 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 called exosomes. Studies on the composition of extracellular vesicles have shown that they can carry a variety of cargoes, including proteins, lipids and nucleic acids, and that this content can vary greatly between cells and conditions, and the specific composition will directly affect the fate and function of extracellular vesicles. Notably, depending on the cell type, extracellular vesicles will display a set of cell type-specific proteins that explain their specific fate and function. Despite the different modes of biogenesis, exosomes and microvesicles show similar appearance, overlapping sizes and generally common composition, which makes it difficult to determine their origin 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 cell restriction factor that prevents HIV-1, Kaposi's sarcoma-associated herpes virus, Ebola virus, and other enveloped viruses from being released from the cell surface. BST2 was originally identified as a type II transmembrane glycoprotein expressed in bone marrow stromal cell lines and involved in the growth of pre-B cells, and is reported to be associated with innate immune responses. Multiple studies have elucidated that aberrant expression of BST2 is found in a variety of cancers, such as breast cancer, oral cancer, lung cancer, 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, and BST2 promotes the migration, invasion, and formation of invadopodia in breast cancer cells. Transcription factor SP1 activates BST2 to accelerate the proliferation and migration of pancreatic cancer cells. However, how BST2 regulates cholangiocarcinoma (CCA) progression remains unstudied. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker BST2 for predicting the prognosis of immunotherapy for cholangiocarcinoma and its application to solve the problems existing in the above-mentioned prior art. The present invention screens out differentially expressed protein molecular markers by mass spectrometry analysis of bile-derived microvesicles, and predicts the prognosis of cholangiocarcinoma patients by detecting the expression level of specific protein molecular markers and comparing them with the reference level. Based on the above research results, the present invention is 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 cholangiocarcinoma.

[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 products.

[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] The present invention found that BST2 can be used as a molecular marker for predicting the prognosis of cholangiocarcinoma, and the overall survival rate of patients with high expression of BST2 in bile microvesicles of cholangiocarcinoma was significantly lower than that of patients with low expression of BST2. The present invention provides a new marker for predicting the prognosis of cholangiocarcinoma and formulating treatment plans, which is of great significance for accurately predicting the prognosis risk of patients and enriching the treatment strategies for cholangiocarcinoma. 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 drawings required for use in the embodiments will be briefly introduced below. 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 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 It is the relative expression amount of BST2 protein in the 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 2 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0024] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, 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 judge the prognosis of patients with cholangiocarcinoma. The present invention finds that BST2 is abnormally highly expressed in the microvesicles secreted by the bile of patients with cholangiocarcinoma, 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 by the bile is negatively correlated with the overall survival of patients with cholangiocarcinoma.

[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 cholangiocarcinoma.

[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 embodiment of the present invention also provides 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, a substance for detecting the expression level of the molecular marker BST2 in bile microvesicles by mass spectrometry or ELISA enzyme-linked immunosorbent assay.

[0031] The embodiment of the present invention also provides a product for predicting and / or diagnosing cholangiocarcinoma, 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 reagents 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 cholangiocarcinoma, and the prognosis is the overall survival of the patients.

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

[0038] The 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 the above-mentioned biomarkers in a sample of a subject, and;

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

[0041] 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 amount of BST2 molecular markers in extracellular microvesicles separated from the bile of bile duct cancer patients is detected to predict the degree of neural invasion tumor in bile duct cancer patients. The treatment plan can be selected and changed according to the prediction, and the survival time and quality of life of patients can be improved, which has 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) Biliary tract cancer is confirmed by puncture combined with imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examination.

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

[0049] (4) Clear clinical data, follow-up information 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 μmol / l, alkaline phosphatase, bile aminotransferase (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 selected, and microvesicles and exosomes in 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 for protein molecules that are significantly differently expressed in the bile of cholangiocarcinoma patients compared to 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) Biliary tract cancer is confirmed by puncture combined with imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examination.

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

[0068] (4) Clear clinical data, follow-up information 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 μmol / l, alkaline phosphatase, bile aminotransferase (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 groups with high and low BST2 expression, 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 , bile microvesicle BST2 was used to screen cholangiocarcinoma, and the area under the ROC curve was 0.776. When the cutoff value was 429.7427, the sensitivity and specificity were 77.3% and 76.9%, respectively. When the detection value was greater than 429.7427, it can be considered that the possibility of nerve invasion in patients with cholangiocarcinoma is high, and further pathological diagnosis should be performed.

[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. The 1-year and 3-year overall survival (OS) rates were also significantly different. Figure 1 Therefore, 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 clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A molecular marker for predicting the prognosis of cholangiocarcinoma immunotherapy, characterized in that: The molecular marker is bone marrow stromal cell antigen 2.

2. Use of the molecular marker as claimed in claim 1 as a detection target in the preparation of a product for predicting and / or diagnosing cholangiocarcinoma.

3. 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 products.

4. A product for predicting and / or diagnosing bile duct cancer, characterized in that: The product uses the molecular marker described in claim 1 as a detection target.

5. The product according to claim 4, characterized in that The product comprises a reagent or a kit.

6. The product according to claim 4 or 5, characterized in that The product uses a blood specimen of a patient to be tested as a sample to be tested.

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