| Abstract|| |
Aims: We aimed to determine whether lymphocyte activation gene 3 (LAG-3), also known as CD223, is associated with microvessel density (MVD) in primary hepatocellular carcinoma (HCC), as well as their clinical significance in predicting survival. Materials and methods: One hundred and twenty-seven patients were enrolled in the study. Samples were obtained on resection at the Department of Hepatobiliary Surgery of the Qingdao Municipal Hospital from June 2014 to June 2016. Immunohistochemistry was used to determine vessel density and LAG-3 abundance. Statistical analyses were performed to test for correlation of LAG-3 density and other clinicopathological variables with overall survival (OS). Results: High LAG-3 abundance was significantly correlated with increased MVD in primary HCC (P < 0.05). The χ2 test revealed a significant association of LAG-3 with preoperative AFP level, tumor diameter, N stage, and the presence of HBV infection (P < 0.05). Patients with high LAG-3 expression had shorter OS compared to those with low LAG-3 expression (P < 0.05). The Cox proportional hazards model showed that both higher LAG-3 and MVD density, age, the number of tumors, preoperative AFP level, tissue differentiation, Child–Pugh grade, and lymph node metastasis correlated with survival. Conclusions: High expression of LAG-3 is associated with angiogenesis and poor prognosis in HCC patients. With the deepening of research, LAG-3 is likely to become a novel biomarker for clinical diagnosis and prognosis and can even be a therapeutic target of HCC.
Keywords: Clinicopathological Features, LAG-3, MVD, primary hepatocellular carcinoma, prognosis
|How to cite this article:|
Tian J, Liu Y, Zhang TL, Xiao YN, Guo CY, Xie YH, An ZJ. Clinical significance of LAG-3 on microvessel density in primary hepatocellular carcinoma. Indian J Pathol Microbiol 2022;65:581-8
|How to cite this URL:|
Tian J, Liu Y, Zhang TL, Xiao YN, Guo CY, Xie YH, An ZJ. Clinical significance of LAG-3 on microvessel density in primary hepatocellular carcinoma. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 15];65:581-8. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/581/351605
| Introduction|| |
Hepatocellular carcinoma (HCC) is a common primary tumor. According to the latest data released by Database of global cancer epidemiology (GLOBOCAN) 2018, the number of newly added HCC cases has reached 841,000 every year in the world. It ranks sixth in the list of malignant tumors, and with 7.82 million deaths recorded every year globally, it ranks second among all causes of cancer deaths in the world. Primary liver cancer caused by heterosexual hepatitis accounts for about 75% to 85%. The incidence and mortality rates of HCC are two to three times higher in males than in females. In contrast to the declining mortality rates of all other common cancers (e.g., breast cancer), the mortality rate of HCC continues to increase at a rate of approximately 2%–3% per year. The development of HCC is associated with hepatitis B or C virus infection, alcohol abuse, nonalcoholic steatohepatitis, consumption of aflatoxin-contaminated food, and schistosomiasis. Recent studies have suggested that smoking, obesity, type 2 diabetes, and drug-induced liver damage are also the risk factors for HCC. Over the past few years, the diagnosis of HCC has relied on ultrasonic monitoring and serological assessment of α-fetoprotein. However, neither the specificity nor the sensitivity of ultrasound and alpha-fetoprotein is sufficient to detect early-onset HCC. So far, the treatment of HCC has also been a challenge. Since the recurrence rate in patients with HCC is as high as 50%–70% 5 years after hepatectomy, reducing the recurrence rate after hepatectomy is the key to improving the overall curative effect of HCC., Therefore, a multidisciplinary approach, individualized comprehensive treatment, and precise treatment of cancer based on genetic information are still the future development directions. According to the recommendations given in Chinese Society of Clinical Ongology (CSCO) 2020 “Guidelines for General Oncology,” the first-line treatment strategies recommended by first-class experts include sorafenib, oxaliplatin-based systemic chemotherapy, lenvatinib, donafenib, atezolizumab, and bevacizumab. At present, immunotherapy with anti-programmed cell death-1 (anti-PD-1), anti-programmed cell death ligand-1 (anti-PD-L1), and anti-CTLA-4 is considered to be the most promising treatment for preventing disease progression and postoperative relapse. Immune checkpoint inhibition blocks negative regulatory signals that directly act on T cells or cells that interact with T cells. Among them, the immune checkpoints of CD8+ T cells have been widely studied as a target of immunotherapy, such as PD-1, lymphocyte activating gene-3 (LAG-3), Tim-3, TIGIT, and CTLA-4. For advanced HCC, several anti-PD-1 monoclonal antibodies (pembrolizumab, nivolumab, camrelizumab), and apatinib have been approved as second-line drugs. However, the efficacy of immunotherapy varies widely among patients with HCC due to resistance or treatment-emergent side effects. Therefore, it is necessary to develop more effective immune checkpoint suppression therapies with limited potential side effects.
As a member of immunoglobulin superfamily, LAG-3 is mainly expressed on the upper surface of activated natural killer (NK) cells, T lymphocytes, B cells, and dendritic cells and binds to major histocompatibility complex class II (MHC-II) molecules, blocks the binding between T-cell antigen receptor (TCR) and MHC-II molecules, and inhibits the activation of T lymphocytes. Therefore, LAG-3 plays a negative role in immunoregulation. It was cloned as a membrane protein for the first time in 1990. In fact, fibrinogen-like protein 1 (FGL-1) is another major functional ligand of LAG-3, which is secreted from hepatocytes and facilitates the mitotic and metabolic functions of target cells. However, blocking the FGL-1–LAG-3 pathway stimulates tumor immunity and inhibits tumor growth. In addition to MHC-II molecules, LAG-3 also binds to Gal-3 and LSECtin, which could explain the inhibitory effect of LAG-3 on various types of lymphocytes without involving MHC-II molecules.,
Angiogenesis plays an important role in the growth, invasion, and metastasis of tumors. Rapid and exponential growth of tumors requires physiological or pathological neovascularization, and angiogenesis is parallel to metastasis. The current gold standard for measuring tumor angiogenesis is microvessel density (MVD), which has been shown to be a predictor of tumor progression and poor prognosis. CD34 is an important endothelial marker that has been shown to be used in the evaluation of angiogenesis. HCC is a highly vascular tumor, which makes it an attractive target for the development of effective anticancer drugs.
It was shown that FGL-1 deletion induced epithelial–mesenchymal transition (EMT) and angiogenesis in LKB-1–mutant lung adenocarcinoma. The expression of MVD in tumor stroma has been shown to be negatively correlated with patient prognosis. Therefore, we designed this experiment to investigate whether LAG-3 expression was associated with MVD and how LAG-3 was related to patient prognosis. Therefore, we hope that we can predict that LAG-3 can be used as an immunotherapy target for HCC just like PD-1.
| Materials and Methods|| |
Patients and tissue specimens
A total of 127 formalin-fixed and paraffin-embedded tumor samples from patients who underwent radical HCC surgery in the East Hospital of Qingdao Municipal Hospital from June 2014 to June 2016 were included in this study. All of them are patients after surgical treatment, and they haven't taken targeted therapy, immunotherapy, neoadjuvant radiotherapy and chemotherapy, or various treatment measures such as tumor ablation and hepatic artery embolization chemotherapy. Besides, they must have a clear pathological diagnosis. Patients who received neoadjuvant therapy that could interfere with the evaluation of LAG-3 density were excluded. Clinical records and histopathologic diagnoses of the patients were fully documented. Tissue samples were collected in accordance with the guidelines of the institutional ethics committee. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional (Qingdao Municipal Hospital) and/or national research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
- Dewaxing and hydration: the dried tissue slices were collected and baked for half an hour in a thermostat at 68 C to melt the paraffin wax on the surface of the slices. The baked sections were removed and dewaxed in xylene solution for 3 minutes each time and 3 times. They were then placed in different levels of ethanol and dehydrated for three times, each lasting for 3 minutes. Then the dehydrated slices were completely washed out with distilled water.
- Antigen thermal repair: preparing a citric acid repair solution with the concentration of 1: 100, putting the treated slices into the citric acid repair solution, heating and pressurizing the slices, continuously heating and pressurizing the slices for 2 minutes and 30 seconds when the temperature of the solution is increased to 100 DEG C, taking the slices out of the citric acid repair solution, putting the slices into room temperature, and washing the slices for three times with a phosphate buffered saline (PBS) solution, wherein the time of each washing lasts for three minutes.
- Hydrogen peroxide sealing: the remaining solutions on the treated slices were sucked dry and placed one by one in a wet box, and then a burette sucked 3% of the hydrogen peroxide solution and uniformly dropped on the slices one by one until the solution could completely cover the tissue slices, the wet box was placed in room temperature for 10 minutes, and then the wet box was rinsed three times with PBS solution, each time for 3 minutes.
- Blocking antigen: the solution remaining on the tissue sections was completely washed out, and then the tissue sections were blotted out with water absorbent paper, and all the processed sections were placed on a wet box; the frozen goat serum was quickly dropped on the sections until the sections were completely covered; and then the wet box was sealed for processing, and the sections were incubated at room temperature for about 30 minutes.
- Instillation of primary antibody: after the above treatment, the serum on the tissue sections was completely blotted out with absorbent paper, the treated tissue sections were spread on the workbench, the primary antibody working solution (prepared with concentrations of LAG-3 and CD34) was dropped on the tissues, and the tissues were sealed in a wet box and incubated in a 4 C refrigerator overnight.
- Instillation of secondary antibody: the sections were put into PBS washing solution to wash the residual liquid, and then washed for three times (3 minutes each time). The sections were spread flat on a wet box and all tissue sections were spiked with the corresponding secondary antibody (enzyme-labeled goat anti-mouse/rabbit IgG polymer). The sections were then immersed for 5 minutes in a dish containing PBS for a total of three times.
- Color development: The sections treated above were taken out, and GENMED diaminobenzidine (DAB) developer was applied dropwise thereto, followed by observation under a microscope and control of the degree of staining of the sections.
- Counterstaining: the sections after staining were fully rinsed with distilled water for 1 minute, then the sections were stained again with hematoxylin solution for 30 seconds, and then the sections were dehydrated with different gradients of ethanol. This process is consistent with the dewatering process used above.
- Dehydration sealing: the counterstained sections were dehydrated with different gradients of ethanol, and after drying, they were placed in a vessel filled with xylene solution and immersed for 3 minutes, a total of 3 times. Drop the gum on the cover glass, seal the cover glass, and take care to prevent the occurrence of air bubbles when dropping the gum. After completing the above operations, place the gum on a glass plate to dry, and write the detailed information of this slide on the glass sheet at the same time, and read the related information of the processed glass sheet under the microscope.
LAG-3 immunohistochemical scoring
Immunohistochemical scoring: All stainings were evaluated independently by at least two pathologists who had no information about the clinical status of the patients (blinded). In cases of discrepancy between the descriptions by the two physicians, re-evaluation was performed by a third physician.
The expression of LAG-3 was evaluated independently by two senior pathologists, both of whom held the position of deputy director or higher using a double-blind method. When the evaluation results of two doctors do not coincide, a third pathologist counts and classifies. Positive staining was determined by the production of yellow or tan granules on the T-lymphocyte membrane in the infiltrated area of the tumor, and the entire field of view of the section was observed under the low-power microscope, followed by ten randomly selected high magnification fields (400×). The expression level was defined by the intensity of positive cell staining and the distribution of positive cells. The intensity of staining was defined as follows: 0 for no staining, 1 for light yellow, 2 for brown, and 3 for tan; the distribution of positive cells was defined as 0 for < 5%, 1 for 5%-25%, 2 for 26%-50%, 3 for 51%-75%, and 4 for > 75%, and the score obtained by multiplying the two results was used as the immunohistochemical score, with 0-1 being negative, 2-4 is weakly positive, 5-8 is moderately positive, and 9-12 is strongly positive. In order to determine whether the expression of LAG-3 is associated with clinical pathological features, the negative and weakly positive expression levels of LAG-3 on the T cell surface were defined as LAG-3 low expression group, and the expression levels were defined as LAG-3 high expression group with medium positivity and strong positivity.Quantification of MVD
The MVD was calculated according to Weidner et al., who first reported the evaluation of MVD in invasive breast cancer and then evaluated the MVD in various solid tumors. The areas with the greatest density of CD34-positive endothelial cells were classified as a “hot spots.” The entire section was scanned at low power (×100) to determine the optimal field for counting. Five random high-power fields were observed under the microscope, and the number of invading cells was counted. Five hot spots were counted for each section and the average was taken.
Patients who underwent curative surgical resection from June 2014 to June 2016 were enrolled in a follow-up study. Follow-up was carried out until June 2020 or death of the patient. All patients had a regular follow-up schedule, including a complete history and physical and radiological examination over the course of the follow-up period. Patients who died due to noncancer-related reasons were excluded.
The statistical package Statistical Package for the Social Sciences (SPSS) version 23.0 was used for data analyses. The χ2 test was carried out to find the correlation between LAG-3 density and increased MVD. It was also used to analyze clinicopathological factors. As the degree of tissue differentiation and Child-Pugh grade of patients belong to grade data, Kruskal-Wallis test was used to test the relationship between LAG-3 expression level and them. Cox proportional risk model is used to determine the influence of each variable on OS. P < 0.05 is statistically significant.All P values were calculated based on two-tailed statistical analyses, and a P value < 0.05 was considered statistically significant.
| Results|| |
LAG-3 expression in HCC
LAG-3 was mainly expressed on the membrane of tumor-infiltrating lymphocytes [Figure 1], [Figure 2], [Figure 3]. The clinicopathological characteristics of the patients are shown in [Table 1]. Out of the 127 HCC tissues cases studied, 58 were defined as LAG-3 poor and 69 were LAG-3 rich. The χ2 test revealed a significant association of LAG-3 with preoperative α-fetoprotein level, tumor diameter, N stage, and the presence of hepatitis B virus (HBV) infection (P < 0.05). There was no significant difference in the other clinicopathological variables observed between LAG-3–poor and LAG-3–rich groups [Table 1].
|Figure 1: LAG-3 staining in HCC tissue (200 × magnification), showing an example of cancer-associated LAG-3–rich group (hematoxylin staining 200×). HCC = hepatocellular carcinoma, LAG-3 = lymphocyte activation gene 3|
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|Figure 2: LAG-3 staining in HCC tissue (200 × magnification), showing an example of cancer-associated LAG-3 moderately rich group (hematoxylin staining 200×). HCC = hepatocellular carcinoma, LAG-3 = lymphocyte activation gene 3|
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|Figure 3: LAG-3 staining in HCC tissue (200 × magnification), showing an example of cancer-associated LAG-3–poor group (hematoxylin staining 200×). HCC = hepatocellular carcinoma, LAG-3 = lymphocyte activation gene 3|
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|Table 1: LAG-3 density and clinicopathological variables (Pearson's χ2 test)|
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CD34 expression in HCC
Please see [Figure 4].
|Figure 4: CD34 staining in intratumoral areas and a typical microvessel structure (hematoxylin stain 100×)|
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|Figure 5: Survival curves for patients with HCC included in the follow-up study. HCC = hepatocellular carcinoma, LAG-3 = lymphocyte activation gene 3. LAG-3 = lymphocyte activation gene 3. LAG-3 = lymphocyte activation gene 3, MVD = microvessel density. HCC = hepatocellular carcinoma, LAG-3 = lymphocyte activation gene 3. CI, confidence interval, HCC = hepatocellular carcinoma, HR = hazard ratio, MVD = microvessel density, OS = overall survival|
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LAG-3 density and MVD
Intratumoral MVD was quantified by measuring CD34-positive staining. Tumors with MVD ≥11 per mm2 area were classified as MVD high (ranging from 2 to 22/mm2; median, 11/mm2). The MVD was significantly higher in LAG-3–rich tumors in comparison to LAG-3–poor tumors (P = 0.011) [Table 2], indicating involvement of LAG-3 in tumor angiogenesis.
Relationship between LAG-3 and prognosis of patients
In the group with high expression of LAG-3, 13 cases were excluded during the follow-up and there were three main reasons for exclusion: 1) loss to follow-up; 2) withdrawal from research, including accidental death, death from other diseases, and temporary change of treatment scheme; and 3) termination: the research time limit expired and the observation was stopped. Eight cases were lost to follow-up, three cases withdrew from the study, and in two cases, the observation was stopped because the time limit of the study had passed. The mean survival time of LAG-3 overexpression was 31.849 months, and the median survival time was 32 months. In the low-expression group of LAG-3, 17 cases were excluded, among which 10 patients were lost to follow-up, four patients dropped out of the study, and in three patients, the observations were stopped because the time limit for the study had expired. The average survival time of patients with low expression of LAG-3 was 42.004 months, and the median survival time was 43 months [Table 3]. Statistical analysis showed that OS in patients with a low expression of LAG-3 was higher than that in patients with a high expression of LAG-3, and the difference was statistically significant (P < 0.005). The results indicated that the prognosis of patients with low expression of LAG-3 was better than that of patients with high expression.
|Table 3: Effect of LAG-3 expression on the survival time of HCC patients Survival curves are shown in Figure 5|
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The Cox proportional hazards model was used to determine the correlation of LAG-3 density and survival rate in 97 patients who completed follow-up [Table 4]. Univariate Cox regression analysis showed that LAG-3 and MVD, age, tumor number, N stage, AFP level before operation, degree of tissue differentiation, and Child–Pugh grade were important prognostic factors affecting OS. Multivariate Cox regression analysis showed that a high density of LAG-3 and MVD, age, tumor number, N stage, AFP level before operation, degree of tissue differentiation, and Child–Pugh grade were independent prognostic factors for survival.
|Table 4: Univariate and multivariate analyses in predicting OS in patients with HCC|
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| Discussion|| |
Although there are many treatments for HCC, such as surgery, radiotherapy, chemotherapy, and targeted therapy, the 5-year survival rate is very low due to its late diagnosis and easy recurrence. Many studies on treatment of liver cancer are related to targeted genes, which have been repeatedly reported as biomarkers for the diagnosis, prognosis, and treatment of cancer patients. Matsuzaki et al. found that in the tissue of Hodgkin's lymphoma (HL) and peripheral blood, the expression of LAG-3 would be higher than that of normal tissue, and it could also inhibit the function of specific anti-tumor CD8+T cells in tumor tissues. If the T cells with positive LAG-3 expression were eliminated, the anti-tumor function of specific CD8+T cells would be restored to a certain extent, and the secretion of cytokines would also be increased. However, few biomarkers have been associated with HCC. At present, the field of oncology believes that the occurrence, development, and metastasis of HCC are not only related to multiple genes, but also closely related to various complex mechanisms such as tumor immune escape in the tumor immune microenvironment. In addition to PD-1 and CTLA-4, other potential immune checkpoints, such as VISTA, OX40, TIM-3, LAG-3, and BTLA, are under investigation. Although there is no report on the efficacy and safety of HCC patients, preclinical studies have proved that LAG-3, TIM-3, and NK cell inhibitory receptors have antitumor activity., This study focuses on the relationship between the LAG-3 gene, which is second only to PD-1 and CTLA-4, and the occurrence, development, and prognosis of HCC, so as to find more effective indicators for the diagnosis, treatment, and prognosis of HCC patients.
In this study, the expression of LAG-3 in the tumor tissues of 127 patients with HCC was found. Noteworthy, the cohort was not balanced for males and females. However, statistical analyses show that LAG-3 density does not depend on gender, so no bias should be introduced. Among the clinicopathological features reported in all studies, the density of LAG-3 was significantly correlated with the preoperative AFP level, tumor diameter, N phase and HBV infection (P < 0.05). The univariate and multivariate analyses of 97 patients followed up showed that LAG-3 and MVD, age, tumor number, N stage, AFP level before operation, degree of tissue differentiation, and Child–Pugh grade are potential independent predictors of HCC survival. LAG-3 and MVD abundance negatively correlated with OS.
In this experiment, we found that LAG-3 is highly expressed in HBV-infected tumor patients, and the literature has revealed that HBV-specific T cells play a key role in the pathogenesis of HCC. However, little is known about the regulation of HBV-specific CD8+ T-cell function in patients with HCC. Liand others evaluated the distribution and function of peripheral blood lymphocytes (PBLs) and HBV-specific CD8+T cells in patients with HCC in a study that showed that the expression of LAG-3 in tumor-infiltrating HBV-specific CD8+ T cells in patients with HCC was significantly up-regulated compared with PBLs and serious functional deficits were identified. This result further supports the possibility that LAG-3 directly and negatively regulates the functional properties of HBV-specific CD8+ T cells in patients with HCC. This suggests that immune checkpoint-blocking therapy may be effective in the treatment of HBV infectious hepatitis.
Previous studies have shown that MVD has been proved to be an independent and important prognostic factor for many different cancers such as breast cancer, gastric cancer, colorectal cancer, and pancreatic cancer. High MVD is related to poor prognosis, which is not studied in this experiment. This experiment has confirmed that high expression of LAG-3 is also an independent factor for poor prognosis. The relationship between neovascularization and canceration was first discovered in 1945. CHABOWSKI M Arghir and others showed that metastatic cancer cells are able to induce angiogenesis before the tumor forms a specific size and that neoangiogenesis is necessary for tumor growth. Up to now, the relationship between LAG-3 and angiogenesis has not been clarified. We measured the expression levels, correlation, and clinical significance of LAG-3 and MVD in human HCC by immunohistochemistry. Our data showed that the expression of LAG-3 was positively correlated with MVD. However, due to the limited laboratory conditions, we were unable to continue to explore the pathways and mechanisms of interaction among the three at a molecular level.
Dysexpression of cancer-related genes is one of the characteristics of cancer. In clinical practice, gene expression detection has been widely used in the diagnosis and treatment of tumors. Wang et al. demonstrated that FGL-1, a protein secreted by the liver, is a major functional ligand of LAG-3 and is independent of MHC-II. FGL-1 inhibits antigen-specific T-cell activation, and excision of mouse FGL-1 promotes T-cell immunity. FGL-1 is highly produced by human cancer cells and its level increases in plasma. FGL-1 in cancer patients is associated with poor prognosis and resistance to anti-PD-1/B7-H1 therapies. Nayeb-Hashemi et al. reported that FGL-1 is a tumor suppressor in HCC and its loss is associated with a poorly differentiated phenotype; they demonstrated that the loss of FGL-1 accelerated the development of HCC. Lack of FGL-1 induced EMT in LKB-1–mutant lung adenocarcinoma. Real-time fluorescence quantitative pcr (RT-qPCR) and Immunohistochemistry (IHC) analyses for angiogenic markers showed that the absence of FGL-1 promoted angiogenesis in LKB-1–mutant lung adenocarcinoma. On the whole, the present study results indicate that deletion of FGL-1 induces EMT and angiogenesis in LKB-1–mutant lung adenocarcinoma. Based on the above results, we speculated that FGL-1–LAG-3 pathway might be one of the complicated mechanisms of angiogenesis in HCC. FGL-1–LAG-3 pathway is a promising target for immunotherapy, which has synergistic effects with PD-1/PD-L1. The combination of anti-LAG-3 monoclonal antibodies and anti-PD-1 monoclonal antibodies has shown exciting effect in tumors which have drug resistance to previous PD-L1 blockers. However, the prognostic significance of FGL-1–LAG-3 pathway and its correlation with PD-L1 in HCC are still unclear.
There are some limitations in the study. First of all, although long-term follow-up increases our findings, we still need to conduct additional prospective studies in a large number of patients to confirm our conclusions. Secondly, the LAG-3 molecule detected by immunohistochemistry is only a sample of the original tumor tissue, which cannot reflect complete information on its interaction with the tumor microenvironment. It is important to note that the number of males and females is not balanced. However, statistical analysis showed that the expression level of LAG-3 is independent of gender, and epidemiology shows that liver cancer is more common in males than in females. However, the mechanism of correlation between LAG-3 expression and other clinicopathological characteristics is still unclear, which would be an important direction of LAG-3 research in the future. Our research has clearly proved the prognostic value of LAG-3 in predicting the survival rate of HCC patients. The reasons for the differences found in different studies are still unclear. In the future, a larger cohort may be needed to determine the positive or negative effects of LAG-3 in the disease process. The trend of blocking immune checkpoints, such as the combination of PD-1/CTLA-4 antibodies and other immunotherapy methods, will require more transformation studies and randomized controlled trials to promote the development of immunotherapy for HCC. Finally, next challenge is to establish and verify the concept of multimode therapy combining immunotherapy with local treatment or surgery.
All authors were involved in the study design and data acquisition, as well as data analysis and drafting of the manuscript. All authors approved the final version of this study. JT, YL, TLZ, and YNX were involved in gathering samples, planning experiments, and performing stainings as well as data analysis (statistics). Furthermore, they all participated in drafting this study. JT, YL, and YNX conducted immunohistochemical stainings and analyzed the results; they also participated in writing this manuscript. YL and CYG planned the study, analyzed the data, drafted the manuscript, and supervised the project.
We thank Xingang Hang and Weiqing Huang (Department of Pathology, Qingdao Municipal Hospital, China) for their expert suggestions and technical assistance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of Gynecology, Qingdao Municipal Hospital, No. 1 JiaoZhaou Road, North District, Qingdao City, Shandong Province
Department of Gynecology, Qingdao Municipal Hospital, No. 1 JiaoZhaou Road, North District, Qingdao City, Shandong Province
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]