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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 12  |  Page : 1188-1192

Expression of programmed death ligand-1 and programmed death 1 in hepatocellular carcinoma and its clinical significance


1 Department of Oncology, Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Jingzhou, Hubei, China
2 Department of Oncology, Caner Hospital, Chinese Academy of Medical Sciences, Chaoyang District, Beijing, China

Date of Web Publication11-Dec-2018

Correspondence Address:
Yan-Hua Xu
Department of Oncology, Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Jingzhou, Hubei
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.204850

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 > Abstract 


Objective: To investigate the correlation between the expression of programmed death 1 (PD-1) and PD ligand-1 (PD-L1) in hepatocellular carcinoma (HCC) and clinical parameters.
Materials and Methods: The study comprised tumor sections from 45 HCC patients treated with curative resection, which were evaluated for PD-1 and PD-L1 protein expression by immunohistochemistry.
Results: PD-1 and PD-L1 expression was increased in cancers compared to adjacent normal tissues, with a positive rate of 37.78% (17/45) and 62.22% (28/45), respectively, which was positively correlated with the tumor stage and lymph node metastasis, negatively with postoperative prognosis. PD-1 positivity was most frequently observed in stromal tumor-infiltrating lymphocytes. The number of PD-1 positive lymphocyte was correlated with PD-L1 positive expression.
Conclusion: PD-L1 and PD-1 are overexpressed in HCC tissues. PD-L1 expression plays a critical role in the pathogenesis of human HCC, suggesting that it might be used as a new biomarker to predict the disease progression and prognosis.

Keywords: Hepatocellular carcinoma, programmed death 1, programmed death ligand-1


How to cite this article:
Long J, Qu T, Pan X F, Tang X, Wan H H, Qiu P, Xu YH. Expression of programmed death ligand-1 and programmed death 1 in hepatocellular carcinoma and its clinical significance. J Can Res Ther 2018;14, Suppl S5:1188-92

How to cite this URL:
Long J, Qu T, Pan X F, Tang X, Wan H H, Qiu P, Xu YH. Expression of programmed death ligand-1 and programmed death 1 in hepatocellular carcinoma and its clinical significance. J Can Res Ther [serial online] 2018 [cited 2019 Nov 22];14:1188-92. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/1188/204850




 > Introduction Top


Hepatocellular carcinoma (HCC) is a very common type of malignant tumor. Because of its severe malignancy and delayed diagnosis, it is a serious threat to human health. Although in recent years, the surgery has been improved continuously, great progresses have been made in neoadjuvant chemotherapy and radiation therapy, prognosis in patients with liver cancer remains poor. Liver carcinogenesis is a complex process, multistep and multiple genes involved, including activation of oncogenes and inactivation of tumor suppressor genes. Immune escape plays an important role in cancer development.[1] Apoptosis and nonresponsiveness of tumor antigen-specific T-cell are the main mechanisms of tumor immune escape, which include costimulatory molecules and corresponding regulation system. The B7 family is very important costimulatory molecules in the immune response including programmed death ligand-1 (PD-L1), also known as B7-H1. The interaction of programmed death 1 (PD-1) with the ligands PD-L1 and PD-L2 inhibits T-cell proliferation. PD-L1 and PD-L2 are mainly expressed on antigen-presenting cells such as DC cells and macrophages. In a common human body, PD-1, as a negative regulation molecule of T-cell proliferation, has an important role in maintaining the immune tolerance. While in tumor, PD-L1 interacts with its receptor PD-1 and then inhibits the proliferation and activation of CD4 and CD8 T-cells. Therefore, they negatively regulate immune responses, mediate tumor immune escape, and then promote tumor growth.[2],[3],[4] This study analyzed the expression of PD-L1 and PD-1 in HCC and paracancerous tissue, the interaction of two molecules and its relationship with clinical characteristics, assessed their impact on tumor progression to provide more evidences for treatment strategies of liver cancer.


 > Materials and Methods Top


Patients and tumor characteristics

Data were collected from previously preserved 45 cases of liver cancer specimens and 15 cases of paracarcinoma tissue samples. It was confirmed histologically that there were not cancer cells in these paracarcinoma tissues. Among these 45 cases of primary HCC, there were 29 cases of cholangiocellular liver cancer and 16 cases of HCC. All samples were provided by the Department of Pathology in our hospital.

The 45 cases of liver cancer specimens were collected by surgery in our hospital from January 2013 to December 2014. These samples were pathologically diagnosed as primary liver cancer. These patients were 38 males and 7 females, aged 35–75 years old and average 53 ± 2.4 years old. The diameter of liver cancer in 26 cases was >5 cm, and that in 19 cases were <5 cm. No envelope was in 20 cases, and lymph node metastasis of porta was in 22 cases. Positive hepatitis B surface antigen was detected in 30 cases, and negative hepatitis B surface antigen was detected in 15 cases. The American Joint Committee on Cancer (2002) Staging System was used to determine liver cancer, 23 cases were Phase I-II and 21 cases were Phase III-IV.[5] All specimens were fixed with 10% neutral formalin, embedded with paraffin, and serial sliced in 4 mm thick. In all 45 cases, there was complete follow-up information by phone and mail. The survival time was calculated from the surgery date to the end date of follow-up (December 2015) or the date of death. Until the end date of follow-up, 15 patients were alive and 30 patients died.

Procedures

Expressions of programmed death 1 and programmed death ligand-1 were determined by immunohistochemistry

The wax of specimen was sliced into the 4 m serial block, after dewaxing and hydration, 3% hydrogen peroxide solution was added at room temperature for 10 min, and rinsed with phosphate-buffered saline (PBS) for 5 min and repeated 3 times. Then, citrate buffer (pH 6.0) was added and cooled to room temperature, after PBS rinsed for 5 min and repeated for 3 times, mouse antihuman PD-1 monoclonal antibody (Sigma-Aldrich, USA. 1:50 dilution) and rabbit antihuman PD-L1 polyclonal antibody (Sigma-Aldrich, USA. 1:50 dilution) were added and incubated overnight at 4°C. After PBS rinsed for 5 min and repeated for 3 times, the ultraView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, USA) was used to visualize the bound primary antibody, developing time was checked under an optical microscope, in 3–5 min, color reaction was stopped by distilled water rinse. The slide was counterstained with hematoxylin for 1 min, rinsed back to blue using water, dehydrated with serial dilutions of alcohol, and sealed with Rhamsan. Immunohistochemical slide was independently assessed by pathologists. Yellow to brown particles appeared in cytoplasm or membrane was considered as a positive chromogenic reaction for PD-L1 and PD-1. PD-L1 expression was determined by semi-quantitative integration method, combined with the intensity and the percentage of positive cells to evaluate the positive expression case.[6] First, whole field of slide was observed under microscope at low magnification, then 5 fields at high magnification (×400) in tumor cell and tumor stroma were selected randomly, strength of staining was graded as no color scores 0, light yellow scores 1, brown yellow scores 2, and brownish brown scores 3; positive cell density was graded as positive cell number ≤10% scores 1, positive cell number 10%–50% scores 2, and positive cell number >50% scores 3. The multiplied result of two scores ≥3 was considered as positive. Moreover, the positive results either in tumor cells or tumor stroma were considered as positive. The positive results of PD-1 were determined using cell counting method. First, the whole field of slide was observed under microscope at low magnification, then higher density of lymphocyte was selected, in which 5 fields at high magnification (×400) were chosen randomly. In each field, PD-1-positive cells among 100 lymphocytes were counted, and its average value was the number of PD-1-positive cells. The average PD-1-positive cells in all cases were decided as threshold values and greater than the threshold was as positive for PD-1 expression, below the threshold was as negative for PD-1 expression.

Statistical analysis

SPSS version 18.0 (IBM, New York, United States) statistical software was used for statistical analysis. The Chi-square test and the Fisher's exact probability test were used to compare the data from various groups. Kaplan–Meier survival curves and log-rank test were used to compare survival rate of groups. P < 0.05 was considered statistically significant.


 > Results Top


Expression of programmed death ligand-1 and programmed death 1 in hepatocellular carcinoma and paracancerous tissue

Immunohistochemistry data showed the positive expression of PD-L1 protein in HCC was 62.22% (28/45), mainly in cytoplasm and membrane of liver cancer cells and tumor stromal lymphocyte. Tumor stromal lymphocyte was small rounded with large nuclear and less pulp, and point distributed. The positive rate of PD-L1 expression on tumor stromal lymphocytes was 48.89% (22/45). The number of tumor-infiltrating lymphocytes with PD-1 positive expression was 0 ~ 50 (12.68 ± 3. 49). The average number 12.68 was set as threshold value, and >12.68 was considered as positive. The positive rate was 37.78% (17/45), which mainly localized in cytoplasm and cell membrane of liver cancer stromal lymphocytes. The Chi-square test found that the expression level of PD-L1 in liver cancer cells and stromal is positively correlated with the infiltration degree of PD-1 positive lymphocytes [P < 0.05, [Table 1]. Moreover, PD-L1 and PD-1 protein was not expressed in 15 cases of paracancer tissue [Figure 1].
Table 1: Relationship between infiltration of programmed death-1 positive lymphocyte and expression of programmed death ligand-1 in tumor cells and stroma of hepatocellular carcinoma

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Figure 1: Immunohistochemical staining in hepatocellular carcinoma of programmed death ligand-1 and programmed death 1: (a) programmed death ligand-1 negative in tumor stromal, (b) programmed death 1 negative in cancer stromal lymphocytes, (c) programmed death ligand-1 negative in tumor, (d) programmed death ligand-1 positive in tumor stromal, (e) programmed death 1 positive in cancer stromal lymphocytes, (f) programmed death ligand-1 positive in tumor

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Expression of programmed death ligand-1 and programmed death 1 in hepatocellular carcinoma and its relation to clinical and pathological characteristics

The expression of PD-L1 in HCC cells was closely related to the tumor node metastasis (TNM) classification of malignant tumors (TNM) (P < 0.05) and lymph node metastases (P < 0.01). Moreover, the expression of PD-L1 in lymphocyte in HCC stroma was also closely related to TNM staging (P < 0.05) and lymph node metastases (P < 0.05). While the expression of PD-1 in HCC showed no significant correlation to clinical and pathological characteristics [Table 2].
Table 2: Relationship between the expression of programmed death ligand-1 and programmed death-1 in hepatocellular carcinoma and clinical and pathological features (n)

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Expression of programmed death ligand-1 and programmed death 1 in hepatocellular carcinoma and its relation with postoperative survival in patients

Kaplan–Meier survival curve analysis of 45 patients showed the expression of PD-L1 protein in cancer cells was associated with the patient's survival. The survival in PD-L1-positive cases was shorter (P < 0.05). PD-1 positive tumor-infiltrating lymphocytes were not related to postoperative survival [Figure 2].
Figure 2: Cancer-specific survival for patients with hepatocellular carcinoma. Data 1: programmed death ligand-1 positive compared to programmed death ligand-1 negative in tumor, respectively (P = 0.025; log-rank), and data 2: programmed death 1 positive compared to programmed death 1 negative in tumor-infiltrating lymphocytes, respectively (P = 0.08; log-rank). PD-L1+ = Programmed death ligand-1 positive, PD-L1− = Programmed death ligand-1 negative, PD-1+: Programmed death 1 positive, PD-1− = Programmed death 1 negative

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 > Discussion Top


PD-1, functioning as an immune checkpoint, belongs to the member of CD28 family. PD-1 is a Type I transmembrane protein, which consists of 268 amino acid, located on PDCD1. It includes an extracellular immunoglobulin variable region (IgV)-like domain followed by a hydrophobic transmembrane domain and an intracellular tail. The intracellular tail contains 2 separate phosphorylation sites, located in immunoreceptor tyrosine-based inhibitory motif, and immunoreceptor tyrosine-based switch motif. PD-1 is mainly expressed in T-cells, B-cells, NK-cells, and membrane surface of a variety of tumor cells.[7]

PD-L1 is a transmembrane protein, belonging to B7 family, and localized to human chromosome 9p24.2. PD-L1 consists of 290 amino acids, extracellular domain containing two Ig constant regions (IgCs) and IgV-like domain, mainly expressed in mature CD4+ T-cell, CD8+ T cell, monocyte, macrophage, B-cell, dendritic cell, and some nonhematopoietic cells such as endothelial cell, islet, and mast cell. Moreover, PD-L1 is overexpressed in a variety of tumor cells.[8],[9]

PD-L1 is involved in immune escape in cancer.[10] Although most of the studies concerned about the expression of PD-L1 in cancer cells, expression and distribution of PD-L1 in tumor-stromal cell were ignored. A few studies reported the expression and distribution of PD-L1 in tumor stromal cells.[11] Wu et al. found that primary liver cancer escaped T-cell-mediated immunity through the expression of PD-L1. This study showed PD-L1 positive expression in tumor-stromal immune cells, and cancer cells were positively correlated with tumor stage and lymph node metastasis, negatively correlated with prognosis. This study also showed that the expression of PD-L1 and PD-1 in HCC was significantly higher than that in paracancer tissue. In this study, the expression of PD-L1 was not correlated with the expression of PD-1. It means that regularity for the expression of PD-L1 and PD-1 in tumor does not exist. It was also found that the expression of PD-L1 in cancer cells had no correlation with the surrounding stromal PD-1 expression, and the expression of PD-1 is not associated with clinical and pathological features of patients. It suggested that PD-L1 still has a biological function when uncoupling with PD-1, then it further hinted there are some receptors other than PD-1 involved in negative immune regulation of PD-L1,[12] but this needs to be confirmed by further studies.

In cancer patients, overexpression of PD-L1 can promote tumor metastasis and lead to increased mortality, so expression of PD-L1 can be used as an indicator of prognosis.[13] Wang et al. found that PD-L1 could be used as an independent indicator of prognosis in patients with pancreatic cancer by analysis of Cox proportional hazards regression model in the study of 81 patients with pancreatic cancer.[14] This study showed that overexpression of PD-L1 in patients with liver cancer was related to relatively late clinical stage and poor prognosis, it may be caused by the lack of normal immune surveillance. Tumor-infiltrating lymphocytes are an important part of the tumor microenvironment. Number and function change of tumor-infiltrating immune cells are valid indicators to assess the overall level of antitumor and determine the postoperative prognosis. Studies found that the expression levels of PD-L1 in ovarian cancer were negatively correlated with the infiltration of CD8 T-cells, and the expression levels of PD-L1 in liver cancer were positively correlated with infiltrating Foxp3 Tregs. This study also found that the expression levels of PD-L1 in hepatic carcinoma were positively correlated with the distribution of PD-1 positive lymphocyte. The liver cancer cells and stromal cells escaped monitoring and destruction of immune system by overexpression of negative costimulatory factors and interaction with ligand PD-L1. This suggests that blocking the PD-L1/PD-1 signaling pathway may be an effective way of cancer immunotherapy and become a new strategy of immune therapy in liver cancer. It needs further study to determine if PD-L1 can become an independent prognostic index of liver cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2001;2:261-8.  Back to cited text no. 3
    
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Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999;5:1365-9.  Back to cited text no. 4
    
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Mamalis A, Garcha M, Jagdeo J. Targeting the PD-1 pathway: A promising future for the treatment of melanoma. Arch Dermatol Res 2014;306:511-9.  Back to cited text no. 7
    
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Hatam LJ, Devoti JA, Rosenthal DW, Lam F, Abramson AL, Steinberg BM, et al. Immune suppression in premalignant respiratory papillomas: Enriched functional CD4+Foxp3+regulatory T cells and PD-1/PD-L1/L2 expression. Clin Cancer Res 2012;18:1925-35.  Back to cited text no. 8
    
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Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, Freeman GJ, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol 2003;33:2706-16.  Back to cited text no. 9
    
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Konishi J, Yamazaki K, Azuma M, Kinoshita I, Dosaka-Akita H, Nishimura M. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res 2004;10:5094-100.  Back to cited text no. 10
    
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Wu K, Kryczek I, Chen L, Zou W, Welling TH. Kupffer cell suppression of CD8+T cells in human hepatocellular carcinoma is mediated by B7-H1/programmed death-1 interactions. Cancer Res 2009;69:8067-75.  Back to cited text no. 11
    
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Kline J, Gajewski TF. Clinical development of mAbs to block the PD1 pathway as an immunotherapy for cancer. Curr Opin Investig Drugs 2010;11:1354-9.  Back to cited text no. 12
    
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Pedoeem A, Azoulay-Alfaguter I, Strazza M, Silverman GJ, Mor A. Programmed death-1 pathway in cancer and autoimmunity. Clin Immunol 2014;153:145-52.  Back to cited text no. 13
    
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Wang L, Ma Q, Chen X, Guo K, Li J, Zhang M. Clinical significance of B7-H1 and B7-1 expressions in pancreatic carcinoma. World J Surg 2010;34:1059-65.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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