|Year : 2018 | Volume
| Issue : 1 | Page : 150-154
Programmed death 1 Ligand 1 expression in breast cancer and its association with patients' clinical parameters
Fei Li1, Yi Ren2, Zhandong Wang1
1 Department of Pathology, Xuzhou Cancer Hospital, Xuzhou 221000, PR China
2 Department of Breast Surgery, Xuzhou Cancer Hospital, Xuzhou 221000, PR China
|Date of Web Publication||8-Mar-2018|
Dr. Zhandong Wang
Department of Pathology, Xuzhou Cancer Hospital, Xuzhou 221000
Source of Support: None, Conflict of Interest: None
Objective: To evaluate the expression of programmed death 1 ligand 1 (PD-L1) in the cancer tissues and tumor-adjacent normal tissues of patients with invasive ductal carcinoma of the breast and to analyze the relationship between the expression of PD-L1 and the clinicopathological features of patients.
Materials and Methods: This study included 112 cases of patients with invasive ductal carcinoma of breast who received surgical treatment from March 2012 to February 2016 in Xuzhou Cancer Hospital. The clinical materials of included patients were retrospectively analyzed. The immunohistochemical assay and real-time polymerase chain reaction (PCR) assay were applied to examine the expression of mRNA and protein of PD-L1 in breast cancer specimens of 112 cases and paired tumor-adjacent tissue specimens of 57 cases. The relationship between PD-L1 protein expression and clinicopathological features of patients was analyzed.
Results: PD-L1 protein was mainly expressed in the cytoplasm. The positive rate of PD-L1 expression in invasive ductal carcinoma was 19.6% (22/112), and the positive rate of PD-L1 expression of tumor-adjacent normal tissues was 3.5% (2/57), indicating that the positive rate of PD-L1 expression of cancerous tissues was significantly higher than that of in tumor-adjacent normal tissues (P < 0.05); the positive expression of PD-L1 was not related with the patients' age, menopause history, family history of breast cancer, tumor size, and location of the tumor (P > 0.05) while it was related with lymph node metastasis, the clinic staging, and histopathological grading (P < 0.05). Real-time PCR was applied to detect the mRNA expression of PD-L1 in breast-invasive ductal carcinoma with the mean ΔCt value of 7.79 ± 2.25. However, the mRNA expression of PD-L1 in normal tumor-adjacent tissues was of low expression with the mean ΔCt value of 12.37 ± 3.33. The difference was statistically significant (P< 0.05).
Conclusion: The expression of PD-L1 in breast-invasive ductal carcinoma was significantly increased, and it was related to histological grading, clinical staging, and lymph node metastasis of breast cancer. PD-L1 may be a significant marker for the prognosis of breast cancer patients.
Keywords: Breast cancer, clinical features, immunohistochemistry, programmed death 1 ligand 1
|How to cite this article:|
Li F, Ren Y, Wang Z. Programmed death 1 Ligand 1 expression in breast cancer and its association with patients' clinical parameters. J Can Res Ther 2018;14:150-4
| > Introduction|| |
Programmed death 1 ligand 1 (PD-L1) is an immunoglobulin superfamily haplotype type I transmembrane glycoprotein, which has been named as a programmed cell death-1 receptor in relation to the apoptosis program. Human PD-1 gene, also known as CD279, was located in the chromosome 2q37.35 with relative molecular weight of 55 kDa and composed of extracellular domain, transmembrane domain, and intracellular domain. PD-L1 was widely expressed on the surface of B lymphocytes, monocytes, natural killer cells, macrophages, and vascular endothelial cells. It was also upregulated in human tumor cell lines, such as ovarian cancer, lymphoma, and malignant melanoma, indicating a close relationship with the occurrence and development of tumors. The expression of PD-L1 in various tissue specimens has been studied, such as colon cancer, malignant melanoma,, nonsmall cell lung cancer, renal cell carcinoma, and esophageal cancer., However, reports on PD-L1 in breast cancer tissue specimens have been few. This study is aimed to investigate the expression of PD-L1 in breast cancer with the more commonly used immunohistochemical technique and real-time polymerase chain reaction (PCR) in clinicopathology and provide references for targeted therapy of breast cancer.
| > Materials and Methods|| |
Data were collected in the pathology department of Xuzhou Cancer Hospital from March 2012 to February 2016. A total of 112 cases of paraffin-embedded specimens with complete patient data were involved, including the histological type of invasive ductal carcinoma. In addition, 57 cases of adjacent normal breast tissues (from the normal breast tissues 5 cm outside the edge of the tumor) were obtained. The inclusion criteria were (1) all cases were of female patients without any treatments of radiotherapy and chemotherapy before the surgery, and (2) patient data was complete. There were 49 cases of tumor with diameter <2 cm and 63 cases of tumor with diameter ≥2 cm; according to histopathological grading, there were 30 cases of grade I and 82 cases of grades II and III. According to AJCC (2002) breast cancer TNM staging standard, there were 68 cases of stages I and II cancer as well as 44 cases of stage III cancer.
Reagents and equipments
Rabbit antihuman PD-L1 polyclonal antibody (Abcam Company, diluted to 1: 200), immunohistochemical staining SP kit and DAB kit (Beijing Zhongshan Golden Bridge Company Biotechnology Co., Ltd.), fixed tissue genomic RNA (centrifugal column) extraction kit (Beijing Bioteke Biotechnology Co., Ltd.), reverse transcriptase (M-MLV RT), DEPC water (Promega), 2× SYBR Green Mix kit (Tiangen Biotech Co., Ltd.), and real-time fluorescence quantitative PCR instrument (ABI 7500) were applied in this study.
The slices of 4 μm were made from paraffin-embedded specimens for immunohistochemical assay. After dewaxing, debenzolization, and hydration, streptavidin anti-biotin protein-peroxidase immunohistochemistry was applied for detection. Specific steps were performed according to the kit instructions. PD-L1 protein expressions in all the specimens were detected. Positive control was set for each staining. For the negative control, PBS was applied instead of the primary antibody.
Real-time polymerase chain reaction assay
Ten slices of 10 μm made from paraffin-embedded specimens were placed into a 1.5 ml centrifuge tube. RNA was extracted through dewaxing, historrhexis, delamination, precipitation, drying, dissolving, and other steps. Specific steps were performed according to the kit instructions. The purity and concentration of total RNA were measured with an ultraviolet spectrophotometer, and the reverse transcription process was performed according to the instructions. The primer sequences for GAPDH were as follows: Upstream 5'-GTGAAGGTCGGAGTCAACG-3', downstream 5'-TGAGGTCAATGAAGGGGTC-3'. The primer sequences for PD-L1 were as follows: Upstream 5'-CTGCATGATCAGCTATGGTGGTG-3', downstream 5'-CAGTTCATGTTCAGAGGTGACTGGA-3'. The circulation conditions were as follows: 95°C predenaturation for 10 s; 95°C denaturation for 5 s, 58°C annealing and extension for 31 s, 40 cycles. A melting curve was set for each reaction.
Determination of immunohistochemical results
The PD-L1 was determined as positive with the appearance of yellow to brownish-yellow particles in the cytoplasm. The expression level of PD-L was evaluated by semi-quantitative integral method, together with the staining intensity and percentage of positive cells. The cases of positive expression (immunohistochemical scores [IHS]) were evaluated, and IHS = a × b. First, the whole field of view of the section was observed with lens at low power, and five high power fields (×400) were randomly selected in cancer cells and carcinoma stroma, respectively. Here, “a” indicated for the intensity of staining, which was graded as follows: No color, a = 0 point; light yellow, a = 1 point; brownish yellow, A = 2 points; dark brown, a = 3 points; and “b” indicated for the percentage of positive cells, which was graded as follows: the number of positive cells ≤10%, b = 1 point; 10%–50%, b = 2 points; >50%, b = 3 points. Two scores were multiplied with each other for obtaining the result. If the result ≥3, it suggested positive expression.
Determination of real-time polymerase chain reaction results
To reduce the sample error, the Ct value of the gene to be measured was corrected. The measures were as follows: The relative copy number of the target gene was obtained by the Ct value of the target gene subtracted by the Ct value of the reference gene GAPDH; ΔCt = Ct of the target gene – Ct of GAPDH; the average value of ΔCt was calculated for each group.
The data were analyzed with SPSS 19.0 statistical software (http://www-01.ibm.com/software/analytics/spss/). The measurement data were expressed by x¯ ± s and compared through t-test. The enumeration data were expressed with a relative number and compared with Chi-square test. Two tails P < 0.05 indicated a statistical difference.
| > Results|| |
Expression of programmed death 1 ligand 1 in invasive ductal carcinoma
Immunohistochemistry was applied to detect the PD-L1 protein, which was mainly expressed in the cytoplasm with the presentation of brownish-yellow particles [Figure 1]. The positive rate of PD-L1 expression was 19.6% (22/112) in invasive ductal carcinoma and 3.5% (2/57) in normal tumor-adjacent tissues. The results showed that the positive rate of PD-L1 expression in cancer tissues was significantly higher than that of tumor-adjacent tissues. The difference was of statistical significance (P< 0.05).
|Figure 1: Expression of programmed death 1 ligand 1 in invasive ductal carcinoma of breast|
Click here to view
Programmed death 1 ligand 1 expression and clinicopathological features of patients
The analysis of PD-L1 protein expression and clinicopathological features of patients showed that PD-L1-positive expression was not related with the patient's age, menopausal history, family history, tumor size, and tumor location (P > 0.05). However, it was related with the clinical stage of patients, lymph node metastasis, and histopathological grade (P< 0.05) [Table 1].
|Table 1: Programmed death 1 ligand 1 expression and clinicopathological features of the included 112 patients|
Click here to view
Programmed death 1 ligand 1 mRNA expression in invasive ductal carcinoma
The high expression of PD-L1 in breast-invasive ductal carcinoma was detected by real-time PCR with an average ΔCt of 7.79 ± 2.25. A low expression in normal tissues was detected with an average ΔCt value of 12.37 ± 3.33, indicating a statistically significant difference (P< 0.05) [Figure 2].
|Figure 2: (a: scatter plot of PD-L1 expression; b: paired expression of PD-L1) Programmed death 1 ligand 1 mRNA expression in invasive ductal carcinoma and tumor-adjacent tissues|
Click here to view
| > Discussion|| |
At present, breast cancer has been the leading female malignancy, and its incidence rate has been increased year after year, leading to a great threat to the health of women., The number of newly found breast cancer cases in the world has been nearly 1.7 million/year, and the number of deaths caused by breast cancer is nearly 600,000/year. In addition, the incidence of breast cancer in developed countries is higher than that of in developing countries. With the advances in medical treatment technology, ever-changing achievements have been obtained in early diagnosis of cancer, surgical treatment, chemotherapy, radiotherapy, and other technologies, resulting in greatly improved prognosis of patients with breast cancer. However, distant metastasis was still observed in about half of the breast cancer patients, leading to failures in treatment. Tumor recurrence and metastasis are the main causes for the death of breast cancer patients.
PD-L1 was an immunoglobulin superfamily haplotype type I transmembrane glycoprotein, which was named as a programmed cell death-1 receptor in relation to the apoptosis program. Ghebeh et al. applied immunohistochemical method to detect the expression of PD-L1 proteins in 44 cases of breast cancer tissues, finding that 34% of tumor epithelium cells were positive, and 41% of tumor-infiltrating lymphocytes were positive. Yanhua et al. applied immunohistochemical method to detect 154 cases of breast cancer tissues, and the positive rate of PD-L1 expression was up to 22.7%. Our results showed that the positive rate of PD-L1 expression was 19.6% (22/112) in 112 cases of breast cancer patients while it was 3.5% (2/57) in normal tumor-adjacent tissues. In our study, the reported positive expression rate of PD-L1 in cancerous tissues was lower than that of reported in previous studies. This finding may be contributed to the different antibodies and PD-L1 positive standard applied in different studies. PD-L1 was also found to be highly expressed in breast-invasive ductal carcinoma tissues, which was associated with histological grading, clinical staging, and lymph node metastasis. The later the clinical stage was and the higher the tumor grade was, the higher the positive rate of PD-L1 expression was. In addition, the positive rate of PD-L1 expression in cancer tissues of patients with lymph node metastasis was higher than that of without metastasis. The results suggested that the positive expression of PD-L1 in breast cancer may be related to tumor progression, and it may be taken as a biomarker for predicting the prognosis of breast cancer patients.
Studies have reported the application of anti-PD-L1 monoclonal antibody in the treatment of tumor-bearing mice. It was observed that the tumor growth of mice was significantly inhibited, and tumor cells were cleared with complete remission in part of the cells. Compared with simple cytotoxic T lymphocytes treatment, the survival rate of tumor-bearing mice could be effectively improved with the treatment combined with the application of cytotoxic T lymphocytes. Thus, PD-1/PD-L1 pathway may be involved in breast cancer subtypes, such as the relapse and metastasis of the triple-negative (negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expressions) breast cancer.
Chemotherapy is a treatment with chemical drugs to prevent the proliferation, invasion, and metastasis of cancer cells until the cancer cells are finally killed. Anthracycline chemotherapeutic drugs have been widely applied in the treatment of breast cancer. Studies showed that adriamycin could effectively reduce the expression of PD-L1 on the surface of breast cancer cells, reduce the binding of PD-L1 to PD-1 on the surface of T cells, and ultimately promote the T cells to specifically kill tumor cells. Studies showed that adriamycin could effectively reduce the expression of PD-L1 on the surface of breast cancer cells, but the expression of PD-L1 was upregulated in the nucleus of breast cancer cells. The reason may be that adriamycin promoted the nuclear migration of PD-L1 on the tumor cell membrane, thereby increasing the expression of PD-L1 in the nucleus of cancer cells. It was also a way for tumor cells to fight against apoptosis when chemotherapeutic drugs were applied for tumor treatment. The pharmacological mechanism of Nimesulide was the selective inhibition of cyclooxygenase II, through the interferon-induced PD-L1 expression on the surface of breast cancer cells, thereby promoting the apoptosis of cancer cells and inhibiting the proliferation and metastasis of cancer cells. However, the selectivity of chemotherapeutic drugs was not strong, and normal cells will be inevitably damaged while killing cancer cells, leading to adverse drug reactions.
This work was supported by Medical science and technology development foundation of Jiangsu University (No.JLY20140116), to Yi Ren.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Grenda A, Krawczyk P. New dancing couple: PD-L1 and microRNA. Scand J Immunol 2017;86:130-4.
Gianchecchi E, Delfino DV, Fierabracci A. Recent insights into the role of the PD-1/PD-L1 pathway in immunological tolerance and autoimmunity. Autoimmun Rev 2013;12:1091-100.
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.
Wang X, Yang L, Huang F, Zhang Q, Liu S, Ma L, et al.
Inflammatory cytokines IL-17 and TNF-α up-regulate PD-L1 expression in human prostate and colon cancer cells. Immunol Lett 2017;184:7-14.
Thierauf J, Veit JA, Affolter A, Bergmann C, Grünow J, Laban S, et al.
Identification and clinical relevance of PD-L1 expression in primary mucosal malignant melanoma of the head and neck. Melanoma Res 2015;25:503-9.
Gadiot J, Hooijkaas AI, Kaiser AD, van Tinteren H, van Boven H, Blank C, et al.
Overall survival and PD-L1 expression in metastasized malignant melanoma. Cancer 2011;117:2192-201.
Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al.
Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nat Med 2002;8:793-800.
Konishi J, Yamazaki K, Azuma M, Kinoshita I, Dosaka-Akita H, Nishimura M, et al.
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.
Mu CY, Huang JA, Chen Y, Chen C, Zhang XG. High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation. Med Oncol 2011;28:682-8.
Donepudi MS, Kondapalli K, Amos SJ, Venkanteshan P. Breast cancer statistics and markers. J Cancer Res Ther 2014;10:506-11.
Tao Z, Shi A, Lu C, Song T, Zhang Z, Zhao J, et al.
Breast cancer: Epidemiology and etiology. Cell Biochem Biophys 2015;72:333-8.
Karagiannis GS, Goswami S, Jones JG, Oktay MH, Condeelis JS. Signatures of breast cancer metastasis at a glance. J Cell Sci 2016;129:1751-8.
Ghebeh H, Mohammed S, Al-Omair A, Qattan A, Lehe C, Al-Qudaihi G, et al.
The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: Correlation with important high-risk prognostic factors. Neoplasia 2006;8:190-8.
Yanhua O, Jun L, Shenqiu L. Expression and significance of programmed death 1 ligand 1 in breast cancer. Guangdong Med J 2015;22:1515-7.
Wang J, Jensen M, Lin Y, Sui X, Chen E, Lindgren CG, et al.
Optimizing adoptive polyclonal T cell immunotherapy of lymphomas, using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains. Hum Gene Ther 2007;18:712-25.
Charo J, Finkelstein SE, Grewal N, Restifo NP, Robbins PF, Rosenberg SA, et al.
Bcl-2 overexpression enhances tumor-specific T-cell survival. Cancer Res 2005;65:2001-8.
Mittendorf EA, Philips AV, Meric-Bernstam F, Qiao N, Wu Y, Harrington S, et al.
PD-L1 expression in triple-negative breast cancer. Cancer Immunol Res 2014;2:361-70.
Hasan A, Ghebeh H, Lehe C, Ahmad R, Dermime S. Therapeutic targeting of B7-H1 in breast cancer. Expert Opin Ther Targets 2011;15:1211-25.
Ghebeh H, Lehe C, Barhoush E, Al-Romaih K, Tulbah A, Al-Alwan M, et al.
Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: Role of B7-H1 as an anti-apoptotic molecule. Breast Cancer Res 2010;12:R48.
Liang M, Yang H, Fu J. Nimesulide inhibits IFN-gamma-induced programmed death-1-ligand 1 surface expression in breast cancer cells by COX-2 and PGE2 independent mechanisms. Cancer Lett 2009;276:47-52.
[Figure 1], [Figure 2]
|This article has been cited by|
||Pilot Study: Immune Checkpoints Polymorphisms in Greek Primary Breast Cancer Patients
| ||Nyanbol Kuol, Xu Yan, Vanessa Barriga, Jimsheena Karakkat, Stamatis Vassilaros, Ioannis Fyssas, Anastasios Tsimpanis, Sarah Fraser, Kulmira Nurgali, Vasso Apostolopoulos |
| ||Biomedicines. 2022; 10(8): 1827 |
|[Pubmed] | [DOI]|
||The expression of programmed death-ligand 1 and its association with histopathological grade, stage of disease, and occurrence of metastasis in breast cancer
| ||Agung Sindu Pranoto, Haryasena Haryasena, Prihantono Prihantono, Septiman Rahman, Daniel Sampepajung, Indra Indra, Salman Ardy Syamsu, Elridho Sampepajung, Berti Julian Nelwan, Muhammad Faruk, Andi Nilawati Usman |
| ||Breast Disease. 2021; 40(s1): S71 |
|[Pubmed] | [DOI]|
||Resistance mechanisms to programmed cell death protein 1 and programmed death ligand 1 inhibitors
| ||Parmida Sadat Pezeshki, Pouya Mahdavi Sharif, Nima Rezaei |
| ||Expert Opinion on Biological Therapy. 2021; : 1 |
|[Pubmed] | [DOI]|
||Clinicopathological and prognostic significance of programmed cell death ligand 1 expression in patients diagnosed with breast cancer: meta-analysis
| ||M G Davey, É J Ryan, M S Davey, A J Lowery, N Miller, M J Kerin |
| ||British Journal of Surgery. 2021; 108(6): 622 |
|[Pubmed] | [DOI]|
||Determining Factors in the Therapeutic Success of Checkpoint Immunotherapies against PD-L1 in Breast Cancer: A Focus on Epithelial-Mesenchymal Transition Activation
| ||Mariana Segovia-Mendoza, Susana Romero-Garcia, Cristina Lemini, Heriberto Prado-Garcia, francesca bianchi |
| ||Journal of Immunology Research. 2021; 2021: 1 |
|[Pubmed] | [DOI]|
||Prognostic Role of PD-L1 Expression in Invasive Breast Cancer: A Systematic Review and Meta-Analysis
| ||Magno Belém Cirqueira, Carolina Rodrigues Mendonça, Matias Noll, Leonardo Ribeiro Soares, Maria Auxiliadora de Paula Carneiro Cysneiros, Regis Resende Paulinelli, Marise Amaral Rebouças Moreira, Ruffo Freitas-Junior |
| ||Cancers. 2021; 13(23): 6090 |
|[Pubmed] | [DOI]|
||In Vitro Examinations of Cell Death Induction and the Immune Phenotype of Cancer Cells Following Radiative-Based Hyperthermia with 915 MHz in Combination with Radiotherapy
| ||Michael Hader, Simon Streit, Andreas Rosin, Thorsten Gerdes, Martin Wadepohl, Sander Bekeschus, Rainer Fietkau, Benjamin Frey, Eberhard Schlücker, Stephan Gekle, Udo S. Gaipl |
| ||Cells. 2021; 10(6): 1436 |
|[Pubmed] | [DOI]|
||Overexpression of an Immune Checkpoint (CD155) in Breast Cancer Associated with Prognostic Significance and Exhausted Tumor-Infiltrating Lymphocytes: A Cohort Study
| ||Yu-Chen Li, Quan Zhou, Qing-Kun Song, Rui-Bin Wang, Shuzhen Lyu, Xiudong Guan, Yan-Jie Zhao, Jiang-Ping Wu |
| ||Journal of Immunology Research. 2020; 2020: 1 |
|[Pubmed] | [DOI]|
||NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer
| ||Ge Qin, Xin Wang, Shubiao Ye, Yizhuo Li, Miao Chen, Shusen Wang, Tao Qin, Changlin Zhang, Yixin Li, Qian Long, Huabin Hu, Dingbo Shi, Jiaping Li, Kai Zhang, Qinglian Zhai, Yanlai Tang, Tiebang Kang, Ping Lan, Fangyun Xie, Jianjun Lu, Wuguo Deng |
| ||Nature Communications. 2020; 11(1) |
|[Pubmed] | [DOI]|
||Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy
| ||Michael Hader, Deniz Pinar Savcigil, Andreas Rosin, Philipp Ponfick, Stephan Gekle, Martin Wadepohl, Sander Bekeschus, Rainer Fietkau, Benjamin Frey, Eberhard Schlücker, Udo S. Gaipl |
| ||Cancers. 2020; 12(5): 1082 |
|[Pubmed] | [DOI]|
||Programmed Cell Death Ligand 1 in Breast Cancer: Technical Aspects, Prognostic Implications, and Predictive Value
| ||Federica Miglietta, Gaia Griguolo, Valentina Guarneri, Maria Vittoria Dieci |
| ||The Oncologist. 2019; 24(11) |
|[Pubmed] | [DOI]|