|Year : 2018 | Volume
| Issue : 7 | Page : 1655-1659
Expression of leukocyte immunoglobulin-like receptor B2 in hepatocellular carcinoma and its clinical significance
Xinwen Li1, Xiaojuan Wei2, Huirong Xu3, Zuowei Sha3, Aiqin Gao2, Yuping Sun2, Juan Li2, Liyou Xu4
1 Department of Oncology, Jinan Central Hospital, Affiliated to Shandong University, Jinan; Department of Infectious Diseases, Zibo Central Hospital, Zibo, Shandong, China
2 Department of Oncology, Jinan Central Hospital, Affiliated to Shandong University, Jinan, Shandong, China
3 Department of Pathology, Zibo Central Hospital, Zibo, Shandong, China
4 Department of Hepatobiliary and Pancreatic Surgery, Jinan Central Hospital, Affiliated to Shandong University, Jinan, Shandong, China
|Date of Web Publication||19-Dec-2018|
Department of Oncology, Jinan Central Hospital, Affiliated to Shandong University, 105 Jiefang Road, Jinan, Shandong
Department of Hepatobiliary and Pancreatic Surgery, Jinan Central Hospital, Affiliated to Shandong University, 105 Jiefang Road, Jinan, Shandong
Source of Support: None, Conflict of Interest: None
Background: Leukocyte immunoglobulin-like receptor B2 (LILRB2) has recently been considered a promising tumor promoter in human cancers.
Materials and Methods: In this study, the expression of LILRB2 was assessed in 82 samples of surgically resected human hepatocellular carcinoma (HCC) tissues using immunohistochemistry (IHC).
Results: LILRB2 was overexpressed in HCC tissues and its expression was positively and significantly correlated with poor prognostic features of HCC patients, including poor cell differentiation, larger primary tumor size, and shorter overall survival. In addition, there was a positive correlation between the expression of LILRB2 and its classical ligand human leukocyte antigen G in human HCC tissues.
Conclusion: LILRB2 might play an important role in HCC progression and correlate with poor prognosis of HCC.
Keywords: Hepatocellular carcinoma, human leukocyte antigen G, leukocyte immunoglobulin-like receptor B2, prognosis
|How to cite this article:|
Li X, Wei X, Xu H, Sha Z, Gao A, Sun Y, Li J, Xu L. Expression of leukocyte immunoglobulin-like receptor B2 in hepatocellular carcinoma and its clinical significance. J Can Res Ther 2018;14:1655-9
|How to cite this URL:|
Li X, Wei X, Xu H, Sha Z, Gao A, Sun Y, Li J, Xu L. Expression of leukocyte immunoglobulin-like receptor B2 in hepatocellular carcinoma and its clinical significance. J Can Res Ther [serial online] 2018 [cited 2019 Dec 6];14:1655-9. Available from: http://www.cancerjournal.net/text.asp?2018/14/7/1655/246102
| > Introduction|| |
Hepatocellular carcinoma (HCC) is the sixth most prevalent malignancy and the second most common cause of cancer-related mortality in the world., About 55% of the world's new HCC cases come from China, due to high prevalence of chronic hepatitis B virus (HBV) infection. Although the effect of comprehensive treatment for HCC based on surgery has been substantially improved, clinical cure rates and long-term survival rates for HCC patients remain low. At present, extensive studies are focusing on searching novel molecular targets for HCC treatment.
Leukocyte immunoglobulin-like receptors subfamily B (LILRB) is a group of inhibitory receptors, which signal through intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) to negatively regulate immune cell activation. Previous studies found that several members of the LILRB family are overexpressed in hematopoietic cancer cells and solid tumor cells and correlate with adverse prognosis, indicating that LILRBs represent potential targets for tumor treatment.,,
LILRB2 is a typical representative of the LILRB family, which is mainly expressed in monocytes, dendritic cells (DCs), and endothelial cells. LILRB2 can induce the tolerogenic phenotype of human DCs, consequently inducing immunosuppressive T cells, and inhibiting T-cell activation and proliferation. LILRB2 has been found to be overexpressed in leukemia, breast cancer, nonsmall cell lung cancer (NSCLC), and pancreatic cancer cells and contributes to tumor progression.,,, Our previous study has proved that LILRB2 is highly expressed in NSCLC cell lines and tissues and promotes tumor progression through the ERK signaling pathway. In addition, we have also shown that the expression of LILRB2 is positively related with human leukocyte antigen G (HLA-G) (the classical ligand for LILRB2) expression in NSCLC and their coexpression in turn is positively related with patients' poor overall survival (OS). However, the expression and clinical significance of LILRB2 in HCC have not yet been reported.
In this study, the protein expression of LILRB2 in 82 human HCC samples was evaluated by immunohistochemistry (IHC), quantified by immunoreactivity score, and correlated with clinicopathological features and survival of patients. In addition, the correlation between expression of LILRB2 and its ligand HLA-G was also analyzed. This is the first report demonstrating the clinical significance of LILRB2 expression in HCC tissues.
| > Materials and Methods|| |
Patients and tissue samples
A total of 82 patients with HCC, who underwent routine surgery at the Zibo Central Hospital from 2013 to 2017, were enrolled in this study. Their diagnosis was made by a pathological examination, and none of the patients received any preoperative treatment before admission. The patients were classified according to the most recent version of the staging system (8th edition). This study was approved by the Institutional Review Board and Ethics Committee and was conducted in accordance with the Declaration of Helsinki of 1975. Written informed consent was obtained from all patients.
A total of 82 HCC along with 64 peritumoral tissues were cut into consecutive 4-μm-thick sections. The sections were deparaffinized in xylene and rehydrated with graded ethanol. Following rehydration, endogenous peroxidase was inactivated with 0.3% hydrogen peroxide. Antigen was retrieved by putting the sections in ethylenediaminetetraacetic acid buffer with heating in a microwave oven for 15 min. After rinsing with phosphate buffered saline, the sections were incubated overnight at 4°C with rabbit anti-LILRB2 antibody (1:200, Sigma-Aldrich, USA) and mouse anti-HLA-G antibody (1:100, Proteintech, China). To detect the primary antibody binding, the sections were incubated with Elivision Plus Polymer Horseradish Peroxidase (Mouse/Rabbit) IHC Kit (Fuzhou Maixin Biotech. Co., China) according to the instructions. Finally, the visualization signal was developed with 3,3'-diaminobenzidine tetrahydrochloride and all sections were then counterstained with hematoxylin. For negative controls, the sections were incubated with normal mouse or rabbit IgG under the same experimental conditions. All the staining results were scored by two independent pathologists. The proportion score represented the estimated fraction of positive tumor cell (0 = none; 1 = >25%; 2 = 25%–75%; and 3 = >75%); the intensity score represented the estimated average staining intensity of positive tumor cell (0 = none; 1 = weak; 2 = intermediate; and 3 = strong); and the overall amount of protein present in each sample was then expressed as the product or total score of the proportion and intensity scores (ranges = 0–9). A total score ≥4 was defined as positive LILRB2 or HLA-G expression level, and a total score <4 as negative expression level.
All statistical analyses were performed using the SPSS 19.0 software (SPSS, Chicago, IL, USA). Correlations between LILRB2 expression and clinicopathological parameters or HLA-G expression were evaluated by Chi-square test. We defined OS time as the period from the date of surgery to the date of death from any cause or the last day of follow-up evaluation. The median follow-up period was 33 months (range: 5–56 months). The last censor date was on May 30, 2018. Survival curves were drawn using the Kaplan–Meier method and compared with log-rank test. P < 0.05 was considered to indicate statistically significant differences between groups.
| > Results|| |
Leukocyte immunoglobulin-like receptor B2 is highly expressed in human hepatocellular carcinoma tissues
The expression levels of LILRB2 protein in 82 human HCC tissue samples were measured by IHC. Positive staining showed brownish yellow-to-brown particles. LILRB2 was mainly expressed in the cytoplasm of HCC cells [Figure 1]a, [Figure 1]b, [Figure 1]c. LILRB2 positive expression could also be found in cancer stromal cells [Figure 1]a, [Figure 1]b, [Figure 1]c. About 70.73% of the HCC cases (58/82) showed positive expression of LILRB2, whereas 29.27% were LILRB2-negative [Figure 1]d. Only 28.13% (18/64) cases showed positive LILRB2 immunoreactivity in the peritumoral tissues [Figure 1]e. Most of peritumoral normal liver tissues had no or weak cytoplasmic staining for LILRB2 [Figure 1]f. A significant difference in the expression level of LILRB2 was found between HCC tissues and peritumoral nontumor tissues (P < 0.01) [Figure 1]g.
|Figure 1: Expression of leukocyte immunoglobulin-like receptor B2 in human hepatocellular carcinoma and peritumoral tissues. Representative images (a-f) of sections subjected to immunohistochemical staining for leukocyte immunoglobulin-like receptor B2. (a) Weak positive expression in hepatocellular carcinoma tissues, (b) Moderate positive expression in hepatocellular carcinoma tissues, (c) Strong positive expression in hepatocellular carcinoma tissues, (d) Negative expression in hepatocellular carcinoma tissues, (e) Positive expression in peritumoral tissues, (f) Negative expression in peritumoral tissues, and (g) Significantly increased leukocyte immunoglobulin-like receptor B2 expression was detected in human hepatocellular carcinoma cancer cells, compared with peritumoral tissues **P < 0.01|
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Leukocyte immunoglobulin-like receptor B2 expression is correlated with gender, tumor size, and tumor cell differentiation
To evaluate the correlation between LILRB2 expression and clinicopathological factors, its expression level was scored as described in the “Materials and methods” section. Twenty-four of 82 (29.27%) HCC cases with immunoreactive score (IRS) 0–3 were grouped as negative LILRB2 expression (LILRB2-negative) and 58 (70.73%) cases with IRS 4–9 were grouped as positive LILRB2 expression (LILRB2-positive). Statistical correlations of LILRB2 expression with clinicopathological factors of HCC patients are shown in [Table 1]. Male, larger tumor size, and poor tumor cell differentiation were significantly and positively correlated with the LILRB2 expression (P = 0.02, P = 0.042, and P = 0.018, respectively). No significant difference in LILRB2 expression level was found in other clinicopathological parameters, including age, primary tumor number, tumor-node-metastasis stage, hepatitis B virus infection, and liver cirrhosis.
|Table 1: Correlations between leukocyte a b immunoglobulin-like receptor B2 expression and clinicopathological parameters of 82 patients with hepatocellular carcinoma|
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Leukocyte immunoglobulin-like receptor B2 expression in human hepatocellular carcinoma tissues is correlated with poor overall survival
To examine the significance of LILRB2 expression in prognosis of HCC patients, OS curves were constructed using the Kaplan–Meier method. The OS of patients with LILRB2 positive expression was much lower than that of the group with LILRB2 negative expression (P = 0.0438) [Figure 2]. The median survival time of LILRB2 positive group was 30 months while that of LILRB2 negative group was 38 months.
|Figure 2: Relationship between leukocyte immunoglobulin-like receptor B2 expression in hepatocellular carcinoma cancer cells and overall survival of patients. Kaplan–Meier survival analysis showed that the survival time of leukocyte immunoglobulin-like receptor B2-positive group (n = 58) was significantly lower than that of the leukocyte immunoglobulin-like receptor B2-negative group (n = 24) (log-rank, P = 0.0438)|
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Association between leukocyte immunoglobulin-like receptor B2 and human leukocyte antigen G expression in human hepatocellular carcinoma tissues
Our previous study showed that there was a significant association between expression of LILRB2 and its classical ligand HLA-G in primary NSCLC tissues. Here, we analyzed the association between LILRB2 and HLA-G expression in HCC tissues. HLA-G was mainly stained in the cancer cell cytoplasm and membrane [Figure 3]a and [Figure 3]b. Positive immunoreactivity for HLA-G was found in 43 HCC tissue samples (52.44%), whereas 47.56% of HCC cases were HLA-G-negative [Figure 3]c and [Figure 3]d. Among LILRB2-positive cases, the positive rate of HLA-G expression was 60.34% (35/58). On the other hand, among the LILRB2-negative cases, the positive rate of HLA-G expression was only 33.3% (8/24). In addition, LILRB2 expression was significantly correlated with HLA-G expression (P = 0.026) [Table 2] in human HCC tissues.
|Figure 3: Immunohistochemical expression of human leukocyte antigen G in human hepatocellular carcinoma tissues. Left images show representative positive (a) and negative (c) sections subjected to immunohistochemical staining for human leukocyte antigen G. The right images (b and d) indicate magnified images of the boxed areas in a and c|
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|Table 2: Correlation between leukocyte immunoglobulin-like receptor B2 and human leukocyte antigen G expression in human primary hepatocellular carcinoma tissues|
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| > Discussion|| |
LILRB2 gene is located in the region of human chromosome 19q13.4, which also contains a number of closely related immunoglobulin (Ig)-like receptor genes such as killer cell Ig-like receptors., LILRB2 is mainly expressed in the myeloid lineage cells. Early studies focused on the role of LILRB2 on DCs and identified LILRB2 as an inhibitory biomarker of DCs., Subsequently, LILRB2 was reported to be aberrantly expressed in various cancer cells including the chronic lymphoblastic leukemia, primary ductal and lobular breast cancer, and lung cancer.,, In addition, LILRB2 is expressed in hematopoietic stem cells and the interaction with one of its ligand angiopoietin-like protein 2 induces monocytes subtype acute myelocytic leukemia growth. To the best of our knowledge, the expression of LILRB2 in HCC has not been reported. In this study, we firstly performed IHC to examine the LILRB2 expression in 82 cases of human HCC tissues along with 64 nontumor tissues. The results showed that LILRB2 protein was overexpressed in the majority of cancer cells compared with that in the surrounding nontumor tissues. Our data are consistent with those of our previous studies in NSCLC. Of the 82 HCC specimens, 70.73% samples demonstrated positive staining for LILRB2 as compared with 28.13% in the peritumoral nontumor tissues. There was a significant statistical difference in LILRB2 expression between human HCC tissues and peritumoral nontumor tissues. Tumor cells are the main source of LILRB2-positive cells in HCC tissues. The aberrant expression of LILRB2 was related with poor tumor cell differentiation, larger primary tumor size, and patients' poor OS. These results indicate the potential roles of LILRB2 in HCC progression, which needs further study to elucidate the underlying mechanism.
Our previous study showed that expression of LILRB2 was positively related with that of its classical ligand HLA-G in NSCLC. HLA-G is a classical MHC-I molecule and is known to play important roles in the maternal-fetal tolerance and has also been proved to promote cancer development through immunosuppression.,, Previous studies have demonstrated that HLA-G is overexpressed in human HCC and is an unfavorable prognostic marker of HCC patients., In this study, we observed that LILRB2 expression was strongly and positively associated with HLA-G expression in human HCC. In NSCLC cell lines, we found that interference/overexpression of LILRB2 provoked down-regulation/up-regulation of HLA-G expression and stimulation of HLA-G fusion protein resulted in increased expression of LILRB2. Taken together, we speculated that LILRB2-HLA-G interaction in NSCLC cells might occur in an autocrine manner. Importantly, the anti-LILRB2 antibody weakened the upregulation of phospho-ERK1/2 expression induced by HLA-G, indicating that the LILRB2-HLA-G interaction plays an important role in promoting tumor growth and metastasis in NSCLC. Whether the interaction between LILRB2 and HLA-G contributes to the progression of HCC is worth further study.
| > Conclusion|| |
In summary, this study demonstrated that LILRB2 is overexpressed in human HCC tissues and is associated with patients' poor survival. LILRB2 may serve as a marker for poor prognosis of HCC. Further molecular, cellular, and animal model studies are necessary to better understand the function of LILRB2 in HCC development.
Financial support and sponsorship
This work was supported by the Project of Jinan Youth Team for Science and Technology Innovation (Grant no. 201805064) and Shandong Province Natural Science Foundation (Grant no. ZR2017PH 076).
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, et al.
Hepatocellular carcinoma. Nat Rev Dis Primers 2016;2:16018.
GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: A systematic analysis for the global burden of disease study 2013. Lancet 2015;385:117-71.
Reeves HL, Zaki MY, Day CP. Hepatocellular carcinoma in obesity, type 2 diabetes, and NAFLD. Dig Dis Sci 2016;61:1234-45.
Brown D, Trowsdale J, Allen R. The LILR family: Modulators of innate and adaptive immune pathways in health and disease. Tissue Antigens 2004;64:215-25.
van der Touw W, Chen HM, Pan PY, Chen SH. LILRB receptor-mediated regulation of myeloid cell maturation and function. Cancer Immunol Immunother 2017;66:1079-87.
Lozano E, Díaz T, Mena MP, Suñe G, Calvo X, Calderón M, et al.
Loss of the immune checkpoint CD85j/LILRB1 on malignant plasma cells contributes to immune escape in multiple myeloma. J Immunol 2018;200:2581-91.
Zurli V, Wimmer G, Cattaneo F, Candi V, Cencini E, Gozzetti A, et al.
Ectopic ILT3 controls BCR-dependent activation of akt in B-cell chronic lymphocytic leukemia. Blood 2017;130:2006-17.
Liu X, Yu X, Xie J, Zhan M, Yu Z, Xie L, et al.
ANGPTL2/LILRB2 signaling promotes the propagation of lung cancer cells. Oncotarget 2015;6:21004-15.
Zheng J, Umikawa M, Cui C, Li J, Chen X, Zhang C, et al.
Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature 2012;485:656-60.
Liu J, Wang L, Gao W, Li L, Cui X, Yang H, et al.
Inhibitory receptor immunoglobulin-like transcript 4 was highly expressed in primary ductal and lobular breast cancer and significantly correlated with IL-10. Diagn Pathol 2014;9:85.
Carbone C, Piro G, Fassan M, Tamburrino A, Mina MM, Zanotto M, et al.
An angiopoietin-like protein 2 autocrine signaling promotes EMT during pancreatic ductal carcinogenesis. Oncotarget 2015;6:13822-34.
Zhang P, Guo X, Li J, Yu S, Wang L, Jiang G, et al.
Immunoglobulin-like transcript 4 promotes tumor progression and metastasis and up-regulates VEGF-C expression via ERK signaling pathway in non-small cell lung cancer. Oncotarget 2015;6:13550-63.
Zhang Y, Zhao J, Qiu L, Zhang P, Li J, Yang D, et al.
Co-expression of ILT4/HLA-G in human non-small cell lung cancer correlates with poor prognosis and ILT4-HLA-G interaction activates ERK signaling. Tumour Biol 2016;37:11187-98.
Amin MB, Greene FL, Edge SB, Compton CC, Gershenwald JE, Brookland RK, et al.
The eighth edition AJCC cancer staging manual: Continuing to build a bridge from a population-based to a more “personalized” approach to cancer staging. CA Cancer J Clin 2017;67:93-9.
Hirayasu K, Arase H. Functional and genetic diversity of leukocyte immunoglobulin-like receptor and implication for disease associations. J Hum Genet 2015;60:703-8.
Wilson MJ, Torkar M, Haude A, Milne S, Jones T, Sheer D, et al.
Plasticity in the organization and sequences of human KIR/ILT gene families. Proc Natl Acad Sci U S A 2000;97:4778-83.
LeMaoult J, Zafaranloo K, Le Danff C, Carosella ED. HLA-G up-regulates ILT2, ILT3, ILT4, and KIR2DL4 in antigen presenting cells, NK cells, and T cells. FASEB J 2005;19:662-4.
Manavalan JS, Rossi PC, Vlad G, Piazza F, Yarilina A, Cortesini R, et al.
High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells. Transpl Immunol 2003;11:245-58.
Colovai AI, Tsao L, Wang S, Lin H, Wang C, Seki T, et al.
Expression of inhibitory receptor ILT3 on neoplastic B cells is associated with lymphoid tissue involvement in chronic lymphocytic leukemia. Cytometry B Clin Cytom 2007;72:354-62.
Sun Y, Liu J, Gao P, Wang Y, Liu C. Expression of ig-like transcript 4 inhibitory receptor in human non-small cell lung cancer. Chest 2008;134:783-8.
Ferreira LM, Meissner TB, Tilburgs T, Strominger JL. HLA-G: At the interface of maternal-fetal tolerance. Trends Immunol 2017;38:272-86.
Rouas-Freiss N, Moreau P, LeMaoult J, Carosella ED. The dual role of HLA-G in cancer. J Immunol Res 2014;2014:359748.
Carosella ED, Ploussard G, LeMaoult J, Desgrandchamps F. A systematic review of immunotherapy in urologic cancer: Evolving roles for targeting of CTLA-4, PD-1/PD-L1, and HLA-G. Eur Urol 2015;68:267-79.
Cai MY, Xu YF, Qiu SJ, Ju MJ, Gao Q, Li YW, et al.
Human leukocyte antigen-G protein expression is an unfavorable prognostic predictor of hepatocellular carcinoma following curative resection. Clin Cancer Res 2009;15:4686-93.
Amiot L, Vu N, Samson M. Biology of the immunomodulatory molecule HLA-G in human liver diseases. J Hepatol 2015;62:1430-7.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]