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

Abstract

The invention discloses a molecular marker APOE for predicting immunotherapy prognosis of bile duct cancer and application of the molecular marker APOE, and belongs to the technical field of biomedicine. The invention finds that APOE can be used as a molecular marker for predicting prognosis of bile duct cancer, and the overall survival rate of high expression of APOE in bile duct cancer serum microvesicles is obviously lower than that of an APOE low expression group. The invention provides a new marker for predicting the prognosis of the bile duct cancer and formulating a treatment scheme, which is of great significance for accurately predicting the prognosis risk of a patient and enriching the treatment strategy of the bile duct cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a molecular marker APOE 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] Apolipoprotein E (APOE) is a major component of plasma lipoproteins and is responsible for cholesterol transport and metabolism. Although the role of APOE in cholesterol homeostasis and its regulatory mechanisms in atherosclerosis and Alzheimer's disease (AD) are well known, the role and mechanism of APOE in cancer remain elusive. APOE expression is significantly upregulated in gastric cancer and is associated with shorter survival. In addition, APOE upregulation is positively correlated with muscle infiltration, making it a potential molecular marker for predicting the degree of infiltration. However, the expression profile and function of APOE in cholangiocarcinoma remain unclear. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker APOE 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 serum-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 apolipoprotein E.

[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 apolipoprotein E 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 APOE can be used as a molecular marker to predict the prognosis of cholangiocarcinoma. The overall survival rate of patients with high expression of APOE in cholangiocarcinoma serum microvesicles was significantly lower than that of the APOE low expression group. 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 strategy of 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 curve diagram of the overall survival (OS) of the serum microvesicle APOE high expression group and low expression group in Example 2 of the present invention.

[0016] Figure 2 The relative expression of APOE protein in the mass spectrometry of bile microvesicles of patients with cholelithiasis and cholangiocarcinoma in Example 1 of the present invention. In the figure, CCA represents cholangiocarcinoma, CBDS represents cholelithiasis, and the same below.

[0017] Figure 3 This is the expression of APOE in the serum microvesicles of patients with cholelithiasis and cholangiocarcinoma in Example 2 of the present invention.

[0018] Figure 4 Figure 3 is the ROC curve of APOE expression level and lymph node metastasis.

[0019] Figure 5 Figure 3 is the ROC curve of APOE expression level and vascular invasion.

[0020] Figure 6 Figure 2 is the ROC curve of APOE expression level and nerve invasion. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] 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.

[0027] An embodiment of the present invention provides a molecular marker for predicting the prognosis of cholangiocarcinoma immunotherapy, wherein the molecular marker is apolipoprotein E.

[0028] Studies have found that APOE is abnormally highly expressed in microvesicles secreted from the serum of patients with cholangiocarcinoma, and the expression level of APOE in the microvesicles is closely correlated with the overall survival of the patients. Specifically, the expression level of APOE in microvesicles secreted from serum is negatively correlated with the overall survival of patients with cholangiocarcinoma.

[0029] 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.

[0030] The embodiments of the present invention also provide the use of a substance for detecting the expression level of the molecular marker apolipoprotein E in peripheral blood serum microvesicles in the preparation of a product.

[0031] 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.

[0032] In some specific embodiments, the substance includes but is not limited to substances for detecting the expression level of the molecular marker APOE in peripheral blood serum microvesicles by mass spectrometry and ELISA enzyme-linked immunosorbent assay.

[0033] 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.

[0034] In some specific embodiments, the product comprises a reagent or a kit.

[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 product includes a reagent for detecting the APOE expression level in peripheral blood serum.

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

[0038] An embodiment of the present invention further provides a kit, which comprises the above-mentioned device.

[0039] The embodiment of the present invention also provides the above-mentioned device and / or kit for use in performing prognosis assessment on patients with cholangiocarcinoma; the prognosis assessment on patients with cholangiocarcinoma includes assessing the overall survival of the subject.

[0040] The embodiment of the present invention further provides a device for evaluating the prognosis of bile duct cancer, comprising:

[0041] 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;

[0042] ii) an evaluation module, comprising determining the prognosis of the subject according to the expression level of the biomarker determined in i).

[0043] The present invention can make a judgment on the early diagnosis of patients with cholangiocarcinoma by detecting the expression level of APOE in extracellular microvesicles isolated from the peripheral blood of patients with cholangiocarcinoma. APOE is highly expressed in extracellular microvesicles isolated from the peripheral blood of patients with cholangiocarcinoma, while lowly expressed in extracellular microvesicles isolated from the peripheral blood of patients with cholelithiasis. In addition, the present invention also finds that APOE can be used as a molecular marker to predict whether lymph node metastasis of cholangiocarcinoma occurs and the degree of metastasis. Patients with cholangiocarcinoma with high expression of APOE show a significant tendency to be more prone to lymph node metastasis compared to patients with cholangiocarcinoma with low expression of APOE; in addition, by comparing the data in the cohort of the present invention, it is found that the expression level of APOE is significantly correlated with vascular invasion and nerve invasion. Through statistical analysis, the present invention finds that APOE can be used to predict lymph node metastasis, vascular invasion and nerve invasion.

[0044] Example 1

[0045] 1. Screening of protein molecules

[0046] 1.1 Sample Source

[0047] Inclusion criteria:

[0048] (1) Male or female patients aged 18-80 years.

[0049] (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.

[0050] (3) ECOG performance score 0-1, Child-Pugh performance score A or B.

[0051] (4) Clear clinical data, follow-up information and survival time, with the survival time after treatment being greater than 3 months.

[0052] Exclusion criteria:

[0053] (1) Hematological examination: WBC<4.0*10 9 / L,HB<80g / L,PLT<75*10 9 / L.

[0054] (2) Coagulation function: international normalized prothrombin time ratio (INR) > 2.3, or prothrombin time (PT) prolonged > 6 seconds.

[0055] (3) Liver function indicators: serum albumin (ALB) <2.8 g / dl, serum total bilirubin (TBIL) >51.3 μmol / l, alkaline phosphatase, serum transaminase (ALT and AST) >5 times the upper limit of normal value.

[0056] (4) Renal function indicators: serum creatinine (CRE)> 1.5 times the upper limit of normal value.

[0057] (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.

[0058] (6) Ascites, hepatic encephalopathy, Gilbert's syndrome, sclerosing cholangitis, etc.

[0059] Blood samples from 12 patients with cholangiocarcinoma and 12 patients without cholangiocarcinoma who visited Qilu Hospital of Shandong University from 2022 to 2023 were selected, and the microvesicles and exosomes in the serum were separated by ultraspeed gradient centrifugation.

[0060] 1.2 Mass spectrometry

[0061] Mass spectrometry is used to detect all protein molecules in the serum extracellular vesicles of step (1), identify existing and newly discovered protein molecules, and screen protein molecules that are significantly differently expressed in the serum of cholangiocarcinoma patients compared to the serum of non-cholangiocarcinoma patients.

[0062] The results showed that the APOE expression level in the cholangiocarcinoma group was significantly higher than that in the non-cholangiocarcinoma group. Figure 2 .

[0063] Example 2

[0064] 1. Detection of the relative expression of APOE in serum extracellular vesicles of patients with cholangiocarcinoma

[0065] Serum specimens from 38 cholangiocarcinoma patients and 38 non-cholangiocarcinoma patients who visited Qilu Hospital of Shandong University from 2022 to 2024 were clinically selected. The relative content of APOE was detected by immunohistochemistry ELISA, and the differences were analyzed.

[0066] The details are as follows:

[0067] 1.1 Sample Source

[0068] Inclusion criteria:

[0069] (1) Male or female patients aged 18-80 years.

[0070] (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.

[0071] (3) ECOG performance score 0-1, Child-Pugh performance score A or B.

[0072] (4) Clear clinical data, follow-up information and survival time, with the survival time after treatment being greater than 3 months.

[0073] Exclusion criteria:

[0074] (1) Hematological examination: WBC<4.0*10 9 / L,HB<80g / L,PLT<75*10 9 / L.

[0075] (2) Coagulation function: international normalized prothrombin time ratio (INR) > 2.3, or prothrombin time (PT) prolonged > 6 seconds.

[0076] (3) Liver function indicators: serum albumin (ALB) <2.8 g / dl, serum total bilirubin (TBIL) >51.3 μmol / l, alkaline phosphatase, serum transaminase (ALT and AST) >5 times the upper limit of normal value.

[0077] (4) Renal function indicators: serum creatinine (CRE)> 1.5 times the upper limit of normal value.

[0078] (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.

[0079] (6) Ascites, hepatic encephalopathy, Gilbert’s syndrome, sclerosing cholangitis, etc.

[0080] 1.2 Determination of APOE expression

[0081] (1) Extracellular vesicles in serum were separated by ultracentrifugation, and total proteins of exosomes and microvesicles were extracted.

[0082] (2) The relative content of APOE in the protein extracted in step (1) was detected by ELISA. The results are shown in Figure 3 .

[0083] 2. Survival analysis

[0084] All samples were divided into high-expression and low-expression APOE 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.

[0085] The results showed that the overall survival rate of the APOE high expression group was significantly lower than that of the APOE low expression group. There were also significant differences in the 1-year and 3-year overall survival (OS). Figure 1 .

[0086] The results showed that the overall survival rate of the APOE high expression group was significantly lower than that of the APOE low expression group. The incidence of lymph node metastasis in the two different groups was also significantly different. The incidence of lymph node metastasis in the APOE high expression group was significantly higher than that in the APOE low expression group. The results are shown in Table 1.

[0087] 3. The evaluation of the relationship between APOE expression level and lymph node metastasis is shown in Table 1.

[0088] Table 1

[0089] APOE low expression group (n=19) APOE high expression group (n=19) Lymph node metastasis 6 17 No lymph node metastasis 13 2

[0090] Chi-square test: P < 0.001, ROC curve and area under the curve are shown in Figure 4 , area under the curve: 0.864, by Figure 4It can be seen that the area under the ROC curve for screening cholangiocarcinoma with serum microvesicle APOE is 0.864. When the cutoff value is 546.847, the sensitivity and specificity are 73.9% and 93.3%, respectively. When the detection value is greater than 546.847, it can be considered that the possibility of lymph node metastasis in patients with cholangiocarcinoma is high, and further pathological diagnosis should be performed.

[0091] 4. The evaluation of the relationship between APOE expression level and vascular invasion is shown in Table 2.

[0092] Table 2

[0093] APOE low expression group (n=19) APOE high expression group (n=19) Vascular invasion 6 16 No vascular invasion 13 3

[0094] Chi-square test: P = 0.008, ROC curve and area under the curve are shown in Figure 5 , area under the curve: 0.756, by Figure 5 It can be seen that the area under the ROC curve for screening cholangiocarcinoma with serum microvesicle APOE is 0.756. When the cutoff value is 488.8587, the sensitivity and specificity are 72.7% and 81.2%, respectively. When the detection value is greater than 488.8587, it can be considered that the possibility of lymph node metastasis in patients with cholangiocarcinoma is high, and further pathological diagnosis should be performed.

[0095] 5. The evaluation of the relationship between APOE expression level and neural invasion is shown in Table 3.

[0096] Table 3

[0097]

[0098]

[0099] Chi-square test: P < 0.001, ROC curve and area under the curve are shown in Figure 6 , area under the curve: 0.833, by Figure 6 It can be seen that the area under the ROC curve for screening cholangiocarcinoma with serum microvesicle APOE is 0.833. When the cutoff value is 566.9125, the sensitivity and specificity are 77.8% and 95%, respectively. When the detection value is greater than 566.9125, it can be considered that the possibility of lymph node metastasis in patients with cholangiocarcinoma is high, and further pathological diagnosis should be performed.

[0100] In summary, serum microvesicle APOE can be used as a molecular marker to predict the prognosis of cholangiocarcinoma.

[0101] 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 apolipoprotein E.

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. Application of a substance for detecting the expression level of the molecular marker apolipoprotein E 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 includes 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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