PD-1 and PD-L1 correlated gene expression profiles and their association with clinical outcomes of breast cancer

As one of the most frequently occurred malignant tumors, breast cancer (BC) remains the leading cause of cancer-related death for females in many countries [1]. Breast cancer arises from multiple genetic factors, environmental alternations and their complicated interactions [2]. According to the status of biomarkers including estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2), patients with breast cancer were classified into groups of luminal A, luminal B, HER-2 positive and triple negative [3]. It has been accepted that different groups of BC patients benefit from corresponding treatment strategy of chemical and hormonal therapy [4].

Although certain therapeutic combinations have been used as standard treatment in clinical management of BC, some BC patients still could not get satisfactory clinical outcomes [5]. The different outcomes of BC patients indicated that other critical factors also determine the final therapeutic effect such as the immune status of the cells [6]. It is well-accepted that immune escape of tumor cells and aberrant human immune surveillance play essential role in carcinogenesis, progression and metastasis of various types of cancer [7]. As for immune escape of cancer cells, the identification of PD-1 (programmed death 1) and PD-L1 (programmed death-ligand 1) axis was one of the most encouraging finding of cancer therapy in recent years [8]. Serving as an immune checkpoint in tumor microenvironment, the antibodies of PD-1/PD-L1 has shown prominent effect in a large number of cancer types [9].

Previously, PD-L1 expression has been reported to be associated with worse prognosis of triple negative breast cancer patients, which counteract effect of tumor-infiltrating lymphocytes (TILs) [10]. Another study of HER2 + invasive BC patients indicated a positive correlation of PD-L1 expression and CD8+ T cells with favorable clinical outcomes [11]. One research of 1318 BC patients in European suggested that PD-1 positive immune cells in triple negative breast cancer correlated with longer disease-free survival, and tumor-infiltrating lymphocytes (TILs) density was remarkably related with PD-1 and PD-L1 expression in immune cells [12]. Most of the findings implied that medicine concerning PD-1 and PD-L1 immune checkpoint might become novel therapeutic strategies for breast cancer.

Although the critical role of immune checkpoint PD-1 and PD-L1 have been widely reported in a number of malignant tumors, the underlying regulating mechanisms in breast cancer is still unclear. In this study, we performed comprehensive analysis of gene expression profiles related to PD-1 and PD-L1 in breast cancer using transcriptome data from TCGA. The correlation of PD-1 and PD-L1 with other immune biomarkers and immune cells infiltration were revealed. Furthermore, the effect of PD-1 and PD-L1 on clinical outcomes of BC was explored to determine their potential as biomarkers for BC patients prognosis.

It is well-accepted that tumor and its microenvironment is a complex unit to complete the aggressive growth and metastasis of cancer cells [14‐16]. Although a number of chemical and radical therapy have been used in clinical practice of cancer treatment, the immune system of human are still believed to be the most fundamental and effective weapon against cancer [17]. Programmed cell death 1 (PD-1) and corresponding ligand (PD-L1) are one of the most critical biological suppressors of cytotoxic immune reaction, the antibody of which has been authorized by FDA in a unprecedented fast term [18, 19]. However, the inhibitor of PD-1/PD-L1 were not effective in every individual, which require the comprehensive understanding of specific mechanisms underlying PD-1/PD-L1 regulation in carcinogenesis [20].

Using data from TCGA, we first unraveled the expression status of PD-1/PD-L1 in different subtypes and clinical stages of BC patients. PD-L1 expression was decreased in stage III-IV compared with stage I-II. On the basis of molecular classification, basal-like BC subtype showed highest expression of PD-1 and ERBB2 subtype BC had highest PD-L1 expression, which suggest that different subtypes possess various PD-1/PD-L1 status. In addition, PD-1 and PD-L1 expression correlated with the recurrence of BC patients, which also confirm the critical role of PD-1/PD-L1 immune checkpoint in BC progression.

We subsequently identified co-expressed genes of PD-1 and PD-L1 by means of WGCNA. A total of 1065 genes correlated with PD-1 and 99 PD-L1 correlated genes were screened. The GO enrichment analysis of PD-1 correlated genes suggested biological process of T cell activation, regulation of lymphocyte activation, regulation of T cell activation and leukocyte migration. In addition, PD-L1 correlated genes demonstrated enrichment including positive regulation of killing of cells of other organism, T cell apoptotic process, positive regulation of tolerance induction and cytolysis. The results of previous studies were in accordance with the GO enrichment of PD-1/PD-L1 related genes in functional modulation of T cell. For instance, one study suggested that during the transition from DCIS to an invasive lesion, the host cytolytic T cells interacted with the tumor and destroy the tumor tissue, leading to an adaptive upregulation of PD-L1 and tumor protection against immune destruction [21]. PD-L1 is expressed by antigen-presenting cells and results in T cell inactivation by interaction with PD-1 on T-cells. It is therefore of great importance to clarify the biological process and pathways we identified for PD-1/PDL1 related genes, by which immunotherapy with PD-1- and PD-L1-targeted monoclonal antibodies might dramatically change the therapeutic and prognostic landscape for cancer.

Immune infiltration of breast cancer determine the immune activation of tumor microenvironment and is related with clinical outcome of patients. According to the correlation analysis of PD-1 and PD-L1 with immune cell populations, PD-1 and PD-L1 were mainly related with Neutrophils and Fibroblasts. It has been reported that PD-1 protein expression significantly correlated with higher TIL abundance, Ki-67 index, basal-like subtypes, and distant metastasis of triple-negative breast cancer (TNBC) [22]. The interaction of PD-1/PD-L1 immune checkpoint with immune infiltration is a promising research direction in the future. The immune checkpoints of CD28, CD80, CD86, CTLA4, INPP5D, INPPL1, CD58, CD27, CD70, HLA-A, CD74 were also analyzed in relation to PD-1/PD-L1 expression. Finally, PD-1 was found to be mainly associated with HLA-A and INPP5D while PD-L1 correlated with CTLA4 and CD27. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) belongs to immunoglobulin superfamily and encodes a protein transmitting inhibitory signal to T cells, the antibody of which also demonstrated favorable outcome in clinical management [23]. The combination usage of PD-1/PD-L1 with these immune checkpoints might become novel therapeutic targets in the future [24].

Survival analysis of PD-1/PD-L1 expression status demonstrated that the expression of PD-1 was associated favorable survival of breast cancer patients while PD-L1 did not suggest significant association with BC prognosis. In a study of 195 triple-negative breast cancer individuals, PD-1 was found to be significantly related with better disease free survival and overall survival. The results of this study also suggested that PD-1 protein expression in TILs, but not PD-L1 in tumor cells, predicted better prognosis in TNBC [22]. Researchers demonstrated that high expression of PDL1 are associated with favorable clinical outcome in 127 primary breast cancer [25]. Another study of HER2+ invasive BC patients indicated a positive correlation of PD-L1 expression and CD8+ T cells with favorable clinical outcomes [11]. On contrary, PD-L1 expression has been reported to be associated with worse prognosis of triple negative breast cancer patients, which counteract effect of tumor-infiltrating lymphocytes [10]. Response of BC patients to neoadjuvant therapy and survival outcome indicated that PD-L1 predicted better rate of pathological complete response (pCR) [26]. In addition, PD-1/PD-L1 correlated genes such as CD5, CD74, CD96 and CD226 were also related with prognosis of BC patients. The combination of these immune markers with PD-1/PD-L1 might improve the prediction and management of BC patients in the future. Until now, whether PD-1/PL-L1 predict prognosis in BC patients was still in debate. The specific association of PD-1/PD-L1 with BC prognosis still required further large-scale investigations to confirm, which would benefit the potential of PD-1/PD-L1 as prognostic biomarker.

In this study, we reported the expression status of PD-1 and PD-L1 in different subtypes of breast cancer. The PD-1/PDL1 correlated gene profiles were described, the enrichment analysis of which focus on biological process including T cell activation, lymphocyte activation, leukocyte migration, T cell apoptotic process, tolerance induction and cytolysis. PD-1/PD-L1 expression also demonstrated relation with immune cells infiltration and multiple immune checkpoints. High PD-1 expression was associated with better survival of breast cancer patients.