A biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and its application

Abstract

The present invention discloses a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and its application, and relates to the field of biomedical technology. The biomarker is TLR4. The present invention has found that TLR4 can be used as a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma, and the overall survival rate of TLR4 with high expression in cholangiocarcinoma tissue is significantly lower than that of the TLR4 low expression group. In addition, the present invention has also found that patients with intrahepatic cholangiocarcinoma with high expression of TLR4 were found to have a higher incidence of lymph node metastasis and a greater number of metastases. At the same time, the present invention has also found that the expression level of TLR4 is significantly correlated with vascular invasion and neural invasion. Through statistical analysis, it was found that TLR4 can be used to predict vascular invasion and neural invasion. The incidence of vascular invasion and neural invasion in patients with intrahepatic cholangiocarcinoma with high expression of TLR4 is also significantly higher than that in patients with low expression of TLR4.

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 intrahepatic bile duct cancer 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. 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 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.

[0003] TLR4 (Toll-like Receptor 4) is a Toll-like receptor that is widely present in the immune system of humans and other mammals. Its main function is to recognize exogenous pathogens, such as bacterial lipopolysaccharide (LPS), and trigger an immune response. In tumor research, the relationship between TLR4 and tumors is complex, and may involve inflammatory response, immune regulation, and expression levels in tumor cells. So far, there has been no report on using TLR4 as a marker to predict the prognosis of cholangiocarcinoma. Summary of the invention

[0004] The purpose of the present invention is to provide a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a new biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma, which is of great significance for accurately predicting the patient's prognostic risk and enriching the treatment strategy of cholangiocarcinoma.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma, wherein the biomarker is TLR4.

[0007] Furthermore, a higher content of the biomarker in the intrahepatic cholangiocarcinoma tissue of a patient with intrahepatic cholangiocarcinoma indicates a worse prognosis of the patient with intrahepatic cholangiocarcinoma.

[0008] The present invention also provides the use of a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue in the preparation of a product for predicting the prognosis of intrahepatic cholangiocarcinoma.

[0009] Furthermore, the product is a reagent or a kit.

[0010] The present invention also provides a product for predicting the prognosis of intrahepatic cholangiocarcinoma, wherein the product comprises a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue.

[0011] Furthermore, the product is a reagent or a kit.

[0012] The present invention also provides the use of a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue in the preparation of a product for predicting the occurrence of lymph node metastasis of intrahepatic cholangiocarcinoma or the degree of its metastasis.

[0013] The present invention also provides the use of a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue in the preparation of a product for predicting the occurrence of vascular invasion of intrahepatic cholangiocarcinoma.

[0014] The present invention also provides the use of a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue in the preparation of a product for predicting the occurrence of nerve invasion of intrahepatic cholangiocarcinoma.

[0015] Furthermore, the product is a reagent or a kit.

[0016] The present invention discloses the following technical effects:

[0017] The present invention has found that TLR4 can be used as a biomarker for predicting the prognosis of intrahepatic cholangiocarcinoma. The overall survival rate of TLR4 with high expression in cholangiocarcinoma tissue is significantly lower than that of the TLR4 low expression group. In addition, the present invention has also found that TLR4 can be used as a biomarker for predicting the occurrence and extent of lymph node metastasis in intrahepatic cholangiocarcinoma. Patients with intrahepatic cholangiocarcinoma with high expression of TLR4 have been found to have a higher incidence of lymph node metastasis and a greater number of metastases, while patients with intrahepatic cholangiocarcinoma with low expression of TLR4 tend to have no lymph node metastasis or a smaller number of metastases. At the same time, the present invention has also found that the expression level of TLR4 is significantly correlated with vascular invasion and neural invasion. Through statistical analysis, it was found that TLR4 can be used to predict vascular invasion and neural invasion. The incidence of vascular invasion and neural invasion in patients with intrahepatic cholangiocarcinoma with high expression of TLR4 is also significantly higher than that in patients with low expression of TLR4.

[0018] The present invention provides a new marker for predicting the prognosis of intrahepatic cholangiocarcinoma and the occurrence and extent of metastasis, which is of great significance for accurately predicting the prognostic risk of patients and enriching the treatment strategy of cholangiocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 The qPCR detection results of TLR4 expression in cancer and adjacent tissues of 23 cases of intrahepatic cholangiocarcinoma in Example 1;

[0021] Figure 2 The figure is a survival curve of the overall survival (OS) of patients with intrahepatic cholangiocarcinoma grouped according to the different TLR4 expression levels in Example 2;

[0022] Figure 3 Receiver operating characteristic curve for TLR4 expression level and lymph node metastasis;

[0023] Figure 4 Receiver operating characteristic curve for TLR4 expression level and vascular invasion;

[0024] Figure 5 Receiver operating characteristic curve for TLR4 expression level and nerve invasion;

[0025] Figure 6 Figure 2 is the relationship between TLR4 expression and the number of lymph node metastases. DETAILED DESCRIPTION

[0026] 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.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment 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. The intermediate value in any stated value or stated range, and each smaller range between 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.

[0028] 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.

[0029] 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 invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] Cancer and paracancerous tissues of 23 patients who underwent radical resection of intrahepatic cholangiocarcinoma in Qilu Hospital of Shandong University in the past three years and retained intact tissues were collected. After extracting mRNA from cancer and paracancerous tissues, qPCR detection of TLR4 was performed. The results are shown in Figure 1 The results showed that the expression level of TLR4 in cholangiocarcinoma tissue was significantly higher than that in adjacent tissues.

[0033] The extraction method of mRNA is as follows:

[0034] 1. Take out the tissue sample from the -80℃ refrigerator and place it in a 5 mL EP tube. Add 500 μL Trizol to immerse the tissue. Grind the tissue (3rd gear) with an electric rotor (30s) until it is crushed (clean the rotor with water after each sample to reduce cross contamination). Transfer it to a 1.5 mL RNase-free EP tube, add 500 μL Trizol, and place it on ice for lysis for 5 min (pre-cool the centrifuge to 4℃ in advance);

[0035] 2. Add 200 μL chloroform (chloroform volume is 1 / 5 of Trizol volume), shake vigorously (milky red), and place on ice for 10 min;

[0036] 3. Place in a pre-cooled centrifuge and centrifuge (4°C, 12000 rpm, 15 min). Three layers can be clearly seen (the upper colorless and transparent aqueous phase is the RNA layer, the thin milky white layer in the middle is the DNA layer, the lower red layer is the protein layer, and large tissue blocks can be seen at the bottom);

[0037] 4. Carefully and slowly aspirate 400 μL of the supernatant and place it in another new 1.5 mL RNase-free EP tube. Add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 15 min.

[0038] 5. Centrifuge (4°C, 12000 rpm, 15 min), white precipitate can be seen;

[0039] 6. Discard the supernatant, add 75% ethanol to each tube, and mix by inverting;

[0040] 7. Centrifuge (4°C, 12000 rpm, 10 min), and a clear white precipitate can be seen;

[0041] 8. After centrifugation, discard the supernatant, put it into the centrifuge for another quick spin, wash away the remaining liquid with a 10 μL pipette, and open the lid to dry;

[0042] 9. Add DEPC water to each tube according to the size of the precipitate, blow and dissolve the RNA, and measure the concentration with an enzyme marker, OD 260 / 280 Controlled at 1.8-2.0 and stored at -80°C for subsequent qPCR detection.

[0043] Example 2

[0044] 1. Source and preparation method of human cholangiocarcinoma tissue chip

[0045] A total of 42 paraffin biopsy specimens of patients with intrahepatic cholangiocarcinoma diagnosed from January 2010 to December 2020 were collected from 6 tertiary-level A hospitals (i.e., Qilu Hospital of Shandong University, Qianfoshan Hospital, Liaocheng People's Hospital, Linyi People's Hospital, Zibo Central Hospital, and Dongying Shengli Oilfield Central Hospital). All these patients were advanced patients who could not undergo radical resection of intrahepatic cholangiocarcinoma. All postoperative pathology of the patients was reconfirmed by at least 2 physicians from the Department of Pathology of Qilu Hospital of Shandong University.

[0046] After the collected paraffin specimens are pathologically located, a puncher is used to make a tissue chip with a diameter of 1.5 mm. Tissue chips use a fine needle puncher to collect small cylindrical tissues, namely tissue cores, from a large number of donor wax blocks, and then arrange them neatly into the recipient wax block to make a tissue chip wax block. Then a slicer is used to slice the wax block, and the slices are transferred to a glass slide to make a tissue chip. In this way, each tissue chip contains all tissue samples. The specific steps are as follows:

[0047] (1) Microarray design: There are 42 samples in total, and one well is used for each biopsy pathology sample, so a total of 42 wells are required. The slides are treated with poly-lysine in advance.

[0048] (2) Collect cases and related wax blocks: Mark representative points in the paraffin specimens based on HE-stained sections to find typical cholangiocarcinoma tumor tissue and prepare for subsequent drilling.

[0049] (3) Preparation of receptor wax block: Pour melted paraffin into the mold and, after cooling, punch holes neatly with a fine needle puncher to make a tissue chip receptor wax block with a pore diameter of 1.5 mm.

[0050] (4) Punching of donor wax block: Based on the pathological localization results, a tissue core is collected from the donor wax block using a fine needle puncher with a hole diameter of 1.5 mm.

[0051] (5) Assembly of tissue chip wax blocks: Transfer the collected tissue cores to the corresponding holes of the recipient wax block and arrange them neatly.

[0052] And number them.

[0053] (6) Preparation of tissue chip wax block: Place the tissue chip wax block assembled in the previous step in a 55°C incubator for 8 min. When the wax block is semi-melted, take it out and cool it at room temperature. After the receptor module and the newly inserted tissue core are dissolved into one, place it in a 4°C refrigerator for later use.

[0054] (7) Preparation of tissue microarray: Precool the tissue microarray wax block on ice for 20 min before slicing, and make appropriate corrections on the microtome until the entire tissue is sliced. Avoid missing points or residual points. Make 4 μm slices quickly and continuously, then transfer the slices to 45°C warm water and wait for them to unfold naturally. Then carefully stick them on a poly-lysine-treated slide and dry them at room temperature. Bake the slices at 60°C for about 3 min and store them in a -20°C refrigerator for later use (they can also be wax-sealed and stored in a 4°C refrigerator for later use).

[0055] (8) Quality inspection: After the tissue chip is prepared, eosin and hematoxylin (HE) staining is used for histological identification before immunohistochemistry to determine whether it is tumor tissue.

[0056] 2. Survival analysis

[0057] The recombinant Anti-TLR4 antibody (ab307664) was used to perform immunohistochemical staining (IHC) on the cholangiocarcinoma chip and score it. According to the score results, the intrahepatic cholangiocarcinoma patients were divided into the TLR4 low expression group and the TLR4 high expression group for survival analysis (Kaplan-Meier). The results are shown in Figure 2 The results showed that the overall survival rate of the TLR4 high expression group was significantly lower than that of the TLR4 low expression group.

[0058] IHC scoring criteria:

[0059] ① Number of positively stained cells: Observe 5 high-power fields (×200) on each section and count the percentage of positive cells. The number of positive cells <5% is 0 points, 5%-25% is 1 point, 26%-50% is 2 points, 51%-75% is 3 points, and 76%-100% is 4 points.

[0060] ② Positive staining intensity: colorless is 0 points, light yellow is 1 point, brownish yellow is 2 points, and brownish brown is 3 points.

[0061] The product of the two scores (the number of positively stained cells and the positive staining intensity) is the positive level: 0 points for negative (-), 1-4 points for weakly positive (+), 5-8 points for positive (++), 9-12 points for strongly positive (+++), negative and weakly positive are taken as the low expression group, and positive and strongly positive are taken as the high expression group.

[0062] 3. Evaluation of the relationship between TLR4 expression level and lymph node metastasis

[0063] Cholangiocarcinoma often metastasizes to lymph nodes through lymphatic vessels, which has a negative impact on patient prognosis. Lymph node metastasis is usually evaluated by imaging and surgical resection specimen examination. Lymph node metastasis requires a more comprehensive treatment strategy, including surgery, chemotherapy and radiotherapy, to improve the treatment effect. In the treatment of cholangiocarcinoma, lymph node metastasis is an important factor to be considered, which has a significant impact on the patient's survival rate and treatment plan selection. Therefore, the present invention specifically evaluates the relationship between the expression level of TLR4 and lymph node metastasis.

[0064] The results showed that the incidence of lymph node metastasis in the TLR4 low expression group and the TLR4 high expression group was significantly different. As shown in Table 1, the incidence of lymph node metastasis in the TLR4 high expression group was significantly higher than that in the TLR4 low expression group. The receiver operating characteristic curve of TLR4 expression level and lymph node metastasis is shown in Figure 3 , the area under the curve is 0.844.

[0065] Table 1 Relationship between TLR4 expression level and lymph node metastasis

[0066]

[0067] Chi-square test: P < 0.001.

[0068] 4. Evaluation of the relationship between TLR4 expression level and vascular invasion

[0069] As shown in Table 2, the incidence of vascular invasion in the TLR4 high expression group was significantly higher than that in the TLR4 low expression group. The receiver operating characteristic curve of TLR4 expression level and vascular invasion is shown in Figure 4 , the area under the curve is 0.805.

[0070] Table 2 Relationship between TLR4 expression level and vascular invasion

[0071]

[0072] Chi-square test: P = 0.0197.

[0073] 5. Evaluation of the relationship between TLR4 expression level and neural invasion

[0074] As shown in Table 3, the incidence of nerve invasion in the TLR4 high expression group was significantly higher than that in the TLR4 low expression group. The receiver operating characteristic curve of TLR4 expression level and nerve invasion is shown in Figure 5 , the area under the curve is 0.760.

[0075] Table 3 Relationship between TLR4 expression level and nerve invasion

[0076]

[0077] Chi-square test: P=0.0009.

[0078] Example 3

[0079] 1. Detection of the relative content of TLR4 in patients with intrahepatic cholangiocarcinoma

[0080] A total of 40 tumor tissues from patients with intrahepatic cholangiocarcinoma and lymph node metastasis were collected clinically, and the relative content of TLR4 was detected by immunohistochemistry to analyze its differences. The details are as follows:

[0081] 1.1 Sample Source

[0082] Inclusion criteria:

[0083] (1) Biliary puncture combined with imaging examinations such as abdominal enhanced CT, MRI or MRCP, laboratory tests and physical examinations confirmed bile duct cancer.

[0084] (2) The patient is in good general condition and has no serious organic lesions in important organs such as the heart, lungs, kidneys, and brain that would affect the implementation of the puncture surgery and postoperative recovery.

[0085] (3) The puncture was successful, there were no serious postoperative complications, the postoperative recovery was smooth, and no secondary surgery related to the primary disease was performed.

[0086] (4) Clear clinical data, postoperative follow-up data and postoperative survival time, with the postoperative survival time being greater than 3 months.

[0087] Exclusion criteria:

[0088] (1) History of upper abdominal surgery.

[0089] (2) Those who have to undergo palliative surgery or have their surgery terminated due to accidents such as massive bleeding or cardiac arrest during the operation.

[0090] (3) Patients who undergo palliative surgery due to the detection of local organ infiltration or extensive abdominal organ metastasis during surgery.

[0091] (4) Patients who died due to surgery-related complications or other non-surgical accidents after surgery, or whose postoperative survival period was less than 3 months.

[0092] 1.2 After pathological positioning of the collected paraffin specimens, a puncher is used to make a tissue chip with a diameter of 1.5 mm. Tissue chips use a fine needle puncher to collect small cylindrical tissues, namely tissue cores, from a large number of donor wax blocks, and then arrange them neatly into the recipient wax block to make a tissue chip wax block. Then use a slicer to slice the wax block, and transfer the slices to a glass slide to make a tissue chip. In this way, each tissue chip contains all tissue samples. The specific steps are as follows:

[0093] (1) Microarray design: A total of 40 samples were collected, and one well was used for each biopsy pathology sample, for a total of 40 wells. The slides were treated with poly-lysine in advance.

[0094] (2) Collect cases and related wax blocks: Mark representative points in the paraffin specimens based on HE-stained sections to find typical cholangiocarcinoma tumor tissue and prepare for subsequent drilling.

[0095] (3) Preparation of receptor wax block: Pour melted paraffin into the mold and, after cooling, punch holes neatly with a fine needle puncher to make a tissue chip receptor wax block with a pore diameter of 1.5 mm.

[0096] (4) Perforation of donor wax block: Based on the pathological localization results, a tissue core is collected from the donor wax block using a fine needle puncher with a hole diameter of 1.5 mm.

[0097] (5) Assembly of tissue chip wax blocks: Transfer the collected tissue cores to the corresponding holes of the recipient wax block and arrange them neatly.

[0098] And number them.

[0099] (6) Preparation of tissue chip wax block: Place the tissue chip wax block assembled in the previous step in a 55°C incubator for about 8 min. When the wax block is in a semi-melted state, take it out and cool it at room temperature. After the receptor module and the newly inserted tissue core are dissolved into one, place it in a 4°C refrigerator for later use.

[0100] (7) Preparation of tissue microarray: Precool the tissue microarray wax block on ice for about 20 minutes before slicing, and make appropriate corrections on the microtome until the entire tissue is sliced. Avoid missing points or residual points. Make 4 μm slices quickly and continuously, then transfer the slices to 45°C warm water and wait for them to unfold naturally. Then carefully stick them on a poly-lysine-treated slide and dry them at room temperature. Bake the slices at 60°C for about 3 minutes, and store them in a -20°C refrigerator for later use. You can also seal them with wax and store them in a 4°C refrigerator for later use.

[0101] (8) Quality inspection: After the tissue chip is prepared, eosin and hematoxylin (HE) staining is used for histological identification before immunohistochemistry to determine whether it is tumor tissue.

[0102] 1.3 The cholangiocarcinoma chip was scored after immunohistochemistry (IHC) staining using recombinant Anti-TLR4 antibody (ab22048).

[0103] IHC score = (percentage of low-expressing cell area × 1) + (percentage of medium-expressing cell area × 2) + (percentage of high-expressing cell area × 3).

[0104] 2. TLR4 predicts the occurrence and extent of lymph node metastasis in cholangiocarcinoma

[0105] According to the above-mentioned TLR4 immunohistochemistry test results, 40 patients with intrahepatic cholangiocarcinoma with lymph node metastasis were divided into a TLR4 high expression group and a TLR4 low expression group according to the same scoring criteria as in Example 1. The medical records of these patients were carefully screened and the number of lymph node metastases was recorded.

[0106] After statistics, the relationship between TLR4 expression and the number of lymph node metastases is as follows Figure 6 As shown, the results showed that the number of lymph node metastases in patients with intrahepatic cholangiocarcinoma with high TLR4 expression was significantly higher than that in patients with intrahepatic cholangiocarcinoma with low TLR4 expression.

[0107] It can be seen that TLR4 can be used as a biomarker to predict the occurrence and extent of lymph node metastasis in intrahepatic cholangiocarcinoma.

[0108] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue in the preparation of a product for predicting the prognosis of intrahepatic cholangiocarcinoma, characterized in that: The higher the content of TLR4 in the intrahepatic cholangiocarcinoma tissue of a patient with intrahepatic cholangiocarcinoma, the worse the prognosis of the patient with intrahepatic cholangiocarcinoma.

2. The use according to claim 1, characterized in that: The product is a reagent or a kit.

3. Use of a reagent for detecting the content of TLR4 in intrahepatic cholangiocarcinoma tissue in the preparation of a product for predicting the occurrence or extent of lymph node metastasis of intrahepatic cholangiocarcinoma.

4. The use according to claim 3, characterized in that: The product is a reagent or a kit.

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