CN112946268A

CN112946268A - Application of HHLA2 as prognosis marker in preparation of liver cancer prognosis prediction kit

Info

Publication number: CN112946268A
Application number: CN202011607719.3A
Authority: CN
Inventors: 郑利民; 陈敏山; 徐以拓; 张耀军; 许静
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-06-11
Anticipated expiration: 2040-12-29
Also published as: CN112946268B

Abstract

The invention discloses application of HHLA2 as a prognostic marker in preparation of a liver cancer prognosis prediction kit. The research of the invention shows that the expression of HHLA2 in the in situ hepatocellular carcinoma can be detected, the expression of HHLA2 in the in situ hepatocellular carcinoma can be objectively, accurately and efficiently detected according to the counting and scoring of the area of the paratuber hotspot, the survival time of a patient can be more effectively predicted, and the invention also provides an immunohistochemical detection kit for prognosis prediction after liver cancer resection, which comprises a first antibody and a second antibody, wherein the first antibody is the antibody of HHLA2, and the second antibody is the second antibody marked by horseradish peroxidase. The kit can detect the expression of HHLA2 of the liver cancer in situ, has simple experimental method, is convenient, efficient, visual and good in repeatability, and can visually and efficiently distinguish the expression level of HHLA2, so that the survival time of a patient can be better predicted.

Description

Application of HHLA2 as prognosis marker in preparation of liver cancer prognosis prediction kit

Technical Field

The invention relates to the technical field of disease prognosis diagnosis, in particular to application of HHLA2 as a prognosis marker in preparation of a liver cancer prognosis prediction kit.

Background

Primary hepatocellular carcinoma (hereinafter, liver cancer) is one of the most common and worst-prognosis malignant tumors in China, and the morbidity and mortality of the primary hepatocellular carcinoma are in the front of the malignant tumors. The most effective method for treating liver cancer at present is surgical resection and liver transplantation; however, most patients are diagnosed at a middle-to-late stage, do not have surgical conditions, and have a high postoperative recurrence rate, resulting in a low overall five-year survival rate of liver cancer. In recent years, a large number of clinical trials prove that the immunotherapy method for blocking the PD-L1/PD-1 co-inhibition signal pathway can prolong the survival of part of patients with advanced tumors (including liver cancer), but whether the monoclonal antibody of PD-1/PD-L1 is effective depends on the expression in the tumor microenvironment, and the drug effect of low-expression patients is still limited. At present, patients expressing PD-L1 have only about 20 percent response rate to PD-1/PD-L1 immune blocking treatment. Therefore, the search for new immune examination targets for improving the detection of the immune checkpoint agent by liver cancer patients becomes a new possibility. B7-CD28 family member human endogenous retrovirus-H long terminal repeat associated protein 2(HHLA2) and PD-L1 are B7 family members, have homology, and are remarkably highly expressed in various solid malignant tumors, so that the human endogenous retrovirus-H long terminal repeat associated protein 2 and PD-L1 are possible to become another important immune checkpoint and a therapeutic target after PD-L1. Observing the expression of human endogenous retrovirus-H long terminal repeat associated protein 2(HHLA2) in lung cancer tissues, analyzing the relation between the expression level and clinical pathological characteristics and prognosis of a patient, and indicating that HHLA2 is highly expressed in the lung cancer tissues to play an important role in the occurrence and development process of the lung cancer, wherein the HHLA2 can be used as an important factor for prognosis evaluation of the lung cancer patients (Zhangqing, Chengjun, Zhengxiao, Wucheng, Wanqi, Huwenwei, Wuchang Ping, Jingting, Wanzhigang, human endogenous retrovirus-H long terminal repeat associated protein 2 in the lung cancer tissues and the clinical significance thereof [ J, China journal of Experimental surgery, 2018,35(11): 2-Buyi 2146 ]). However, no research report about HHLA2 in prognosis survival prediction of primary hepatocellular carcinoma is found at present.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides application of HHLA2 as a prognostic marker in preparation of a liver cancer prognosis prediction kit.

The second purpose of the invention is to use the combination of the HHLA2 antibody as the first antibody in preparing an immunohistochemical detection kit for prognosis after liver cancer resection.

The third purpose of the invention is to provide an immunohistochemical detection kit for prognosis prediction after liver cancer resection.

The above object of the present invention is achieved by the following technical solutions:

the research of the invention shows that the expression of HHLA2 in the in-situ hepatocellular carcinoma can be objectively, accurately and efficiently detected according to the counting and scoring of the cancer parathermal point region, the survival time of a patient can be more effectively predicted, and more powerful support is provided for effective diagnosis and treatment, so that a doctor is helped to select a patient population and a method more suitable for treatment, and more accurate personalized treatment is carried out. Therefore, HHLA2 is used as a prognostic marker in the application of preparing a liver cancer prognosis prediction kit.

Preferably, the prognosis of liver cancer is prognosis of the life cycle of a patient after hepatoma resection.

Preferably, the kit is an immunohistochemical detection kit.

The invention also provides application of the combination of the HHLA2 antibody as a first antibody in preparing an immunohistochemical detection kit for prognosis after liver cancer resection.

The invention also provides an immunohistochemical detection kit for prognosis prediction after liver cancer resection, which comprises a first antibody and a second antibody, wherein the first antibody is HHLA2 antibody.

Preferably, the second antibody is a horseradish peroxidase-labeled second antibody.

Preferably, the kit further comprises reagents required for immunohistochemical detection.

The invention also provides a method for predicting the prognosis survival period after the hepatoma resection by using the kit, which comprises the following steps:

first, HHLA2 expression in paraffin-sectioned tumor regions of liver cancer resected patients was examined using immunohistochemistry-based techniques. Then, HHLA2 expression was analyzed by advanced inverted microscope in the paraneoplastic region, and the threshold was determined by the minimum p-value method. If the expression level is larger than or equal to the threshold value, the expression level of HHLA2 is high, otherwise the expression level is low, the survival time of the patient with high expression HHLA2 is short in prognosis, and the survival time of the patient with low expression HHLA2 is relatively long.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a new application of HHLA2 as a prognosis marker in liver cancer prognosis prediction.

(2) The detection kit provided by the invention adopts an immunohistochemical method to detect the expression of the in-situ liver cancer HHLA2, has a simple experimental method, is convenient, efficient, visual and good in repeatability, and can visually and efficiently distinguish the level of the expression of the HHLA2, so that the survival time of a patient can be better predicted.

Drawings

FIG. 1 is a graph showing immunohistochemical detection of expression of HHLA2 in different regions of hepatocellular carcinoma.

FIG. 2 is a graph showing the relationship between the expression of HHLA2 in the paracellular carcinoma region and the survival time of patients. A is the relationship between HHLA2 expression and overall patient survival, and B is the relationship between HHLA2 expression and tumor-free survival of patients.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1

An immunohistochemical method is adopted to detect the expression of HHLA2 in hepatocellular carcinoma in situ, so that the expression level of HHLA2 of different hepatocellular carcinoma patients can be better distinguished, and more accurate personalized treatment can be carried out. For the purposes of the above experiment, the first antibody protein molecule for immunohistochemistry was HHLA2, a horseradish peroxidase-labeled secondary antibody.

The specific use steps are as follows:

1. specimen detection

(1) Selecting a liver cancer paraffin section containing a tumor area, and ensuring no large necrosis;

(2) obtaining 8 paraffin slices with the size of 4 microns, baking the slices at the temperature of 60 ℃ for 2 hours, taking out the slices, and slightly cooling the slices;

(3) dewaxing with xylene at room temperature for 2 times, each time for 10 minutes;

(4) washing xylene in 100% ethanol, and sequentially passing through 95% ethanol, 80% ethanol and 70% ethanol for 5 min each time;

(5) washing with double distilled water for 5 minutes;

(6) with 0.3% H₂O₂Blocking endogenous peroxidase activity for 10 minutes at room temperature;

(7) washing with double distilled water for 5 min for 4 times;

(8) antigen retrieval: 10mM citric acid buffer (pH6.0), and autoclaving for 10 min;

(9) naturally cooling for 30 minutes at room temperature;

(10) the water on the back side of the slide and around the tissue was blotted with paper, the sample carefully marked with an immunohistochemical marker pen, washed four times with PBS, 3 minutes each

(11) First antibody HHLA2 (purchased from Abcam under cat # ab214327) was added dropwise to the tissue sections and incubated for 1 hour at room temperature;

(12) PBS was washed four times for 3 minutes each;

(13) adding horseradish peroxidase labeled secondary antibody (purchased from Dako company, K5007 in a cargo number) dropwise, and incubating for 30 minutes at room temperature;

(14) PBS was washed four times for 3 minutes each;

(15) preparing DAB staining solution (solution B: solution C is 1:50), dripping about 50 mu l of the DAB staining solution into each slice, and placing single distilled water to stop color development when a low background is observed and a specific signal appears;

(16) staining with hematoxylin for about 40 seconds, soaking in water to stop staining, and washing with PBS for 3 times (3 min each time);

(17) and baking the mixture in a dry oven at 37 ℃ for about 15 minutes to obtain the neutral environment-friendly sealing piece.

2. Image acquisition

(1) 3 pictures of the paracancer hot spot region are obtained under a high-power visual field (40 times) of an advanced inverted microscope;

(2) the number of signals from HHLA2 positive cells was counted manually, and the final result was the average of 3 photographs (see FIG. 1).

3. Analysis of results

(1) Dividing the analysis result into two groups of cancer paracell expression and non-expression HHLA2 according to a minimum p value method, namely a threshold value of 3.74 in the case;

(2) the number of cells expressing HHLA2 was recorded as "1" (high) for the case where the number of cells was 3.74 or more, and as "0" (low) for the case where the number was not less than 3;

(3) survival analysis showed significant differences in overall survival (P <0.05) for the different cohorts of patients. Finally, in 206 patients who had received primary liver cancer resection, a significant difference in the overall survival (P <0.05) was obtained in both the high and low population of HHLA2 cells by survival analysis (fig. 2), indicating that the prognosis of survival in patients can be effectively predicted by detecting HHLA2 expression in situ hepatocellular carcinoma.

Claims

Application of HHLA2 as a prognostic marker in preparation of a liver cancer prognosis prediction kit.
2. The use of claim 1, wherein the prognosis of liver cancer is prognosis of survival of a patient after hepatoresection.
3. The use of claim 1, wherein the kit is an immunohistochemical detection kit.
Use of a combination of antibodies to HHLA2 as a primary antibody in the preparation of an immunohistochemical detection kit for the prognosis after hepatoma resection.
5. An immunohistochemical detection kit for prognosis prediction after liver cancer resection, which comprises a first antibody and a second antibody, and is characterized in that the first antibody is HHLA2 antibody.
6. The immunohistochemical detection kit according to claim 5, wherein the second antibody is horseradish peroxidase-labeled second antibody.
7. The immunohistochemical detection kit according to claim 6, further comprising reagents required for immunohistochemical detection.