Breast cancer is characterized by a microenvironment often infiltrated by immune cells, including tumor-infiltrating T cells (TILs) and to a lesser extent, NK cells (25). Metastatic dissemination of cancer cells consists of an interplay between cancer cell-intrinsic factors (genetic and epigenetic diversification) and micro environmental immunosuppressive determinants, such as metabolic, stromal and immunological factors. Interindividual variability in the NK receptors repertoire is also influenced by KIR genes, HLA class I alleles and FCGR3A (CD16A) genetic polymorphisms. The presence of steroid hormone receptors, including ER (estradiol E2), mediates immunostimulation or immunosuppression, particularly on NK cells in a time-dependent manner (26–28).
To date, the analysis of gene polymorphisms at the nucleotide level of the HLA-C locus, in combination with those of FCGR3A in breast cancer, hasn’t been rated yet. For this purpose, we carried out a case-control study between BC patients and healthy controls in order to evaluate the impact of the KIR/HLA-C and FCGR3A genes on the pathogenesis and progression of breast cancer in the Italian population and correlate them with proliferation and some key clinical features of BC, including tumor stage and recurrence.
Based on our observations, first of all the absence of HLA-C*05:01:01 allele could be considered protective toward BC, while the presence of C*07:02:01 could play a role in BC progression. We can speculate that a differential expression of these alleles might influence the efficacy of the immune response toward cancer cells given that, as suggested by previous studies (29), different expression of HLA-C7 and HLA-C5 antigens were found. Low levels of HLA-C*07 allele cell surface expression are due to a more restrictive peptide-binding pocket than the HLA-C*05 allele, that has a flatter cleft that allows binding of a larger range of peptides. This situation can stabilize the HLA-C molecule affecting its expression level on the cell surface. As a consequence, the HLA-C7 and HLA-C5 antigens may negatively and positively regulate T cell immunosurveillance, respectively. Also the predisposing effect of the HLA-C*04:01:01 allele to lymph node metastasis could be due to its low cell surface expression (30). These features might cause NK hyporesponsiveness predisposing to tumor invasiveness.
Defective NK cell cytotoxicity has been described in a variety of solid tumors, including breast cancer (25, 31) which was associated with increased frequency of CD56bright NK cells in peripheral blood (32). Thus, while the interaction between inhibitory KIRs and their HLA class I ligands, by the process of “licensing”, allows NK cells to acquire full effector functions, a mismatch makes NK cells hypo-responsive. Also the activating KIR-HLA class I licensing may influence NK unresponsiveness to cancer cells. NK cells expressing activating KIR2DS1 in presence of self-HLA-C2 ligands are poorly responsive toward cancer (33). Our study indicates that in BC patients KIR2DS4ins and KIR2DL2 genes, not licensed by their HLA-C1 ligands, seem to be protective from the neoplasm onset. Conversely, the absence of both KIR2DS4 and KIR2L2 genes may increase the risk of BC occurrence. The protective role of KIR2DS4ins could be due to its inhibitory action on NK response towards bacterial or viral infections, predisposing to a particular tumor microenvironment. Indeed, it’s known that KIR2DS4ins receptor recognizes recA peptides derived by pathogens, mainly in the context of HLA-C5 antigen (34). Also, the protective role on BC pathogenesis of the inhibitory KIR2DL2 receptor, unlicensed by C1 ligands, maybe due to the NK cell unresponsiveness to cancer cells. Our study evidenced that a simultaneous absence of the inhibiting KIR2DL2, not licensed by its cognate ligand, and the KIR2DS4ins gene increases the risk of tumor progression nine-fold.
Furthermore, we can also speculate that the activating KIR2DS1 might play a role in breast cancer aggressiveness, maybe supporting a chronic over-stimulation of NK cells. The association of this gene with increased BC risk had already been highlighted in another analysis on Turkish patients with advanced BC compared to controls (35). This correlation might depend by the influence in patients with advanced BC from the simultaneous presence of its equivalent inhibitory, KIR2DL1 gene. On the contrary, the presence of KIR2DL1 in the absence of the KIR2DS1 counterpart, in advanced tumors, suggests a putative “antitumor role” of the KIR2DL1 receptor. This information is also supported by analysis of Ashoury E (36). In addition, interestingly, BC patients with lymph node metastasis have shown a significantly higher frequency of the KIR2DL2 gene in presence of HLA-C1/C1 (OR = 4.25) than healthy controls, that by the effect of the process of “licensing” could favor full pro-inflammatory effector functions.
We could speculate that the activation of inflammation mediated by KIR2DS1 gene could be turned off by KIR2DL2/KIR2DS4, but only when they are not licensed by the HLA-C1 ligand. Such results suggest that the susceptibility to BC transformation and progression might depend on the type of genotype combinations. Indeed, the involvement of activating KIRs in cancer pathogenesis was also observed by other Authors (37, 38) in chronic myeloid leukemia (CML) and nasopharyngeal carcinoma, respectively. Increases in the levels of innate immune response stimulation may contribute to an increased risk of some virus-associated cancers, maybe through an amplified inflammatory response triggered by NK cells (or other effector cells) expressing activating KIRs.
Examining the influence of KIR genes on lymph node status, in the context of hormone markers, we did not evidence any difference in the progression of disease between ER or PR positive patients, even though in these patients a prevalent frequency of the KIR2DL3 gene and a reduction of KIR2DS4ins gene were specifically detected. It is possible to hypothesize that breast cancer hormones may influence in different way some immune responses depending on genetic background (i.e. every HER-2+ BC patients in our study carried the KIR2DS1 gene and showed signs of metastasis).
Fcγ receptors are essential for the ADCC pathway and FCGR3A gene functional polymorphisms may affect the killing function of immune effector cells. FCGR3A-158 G/T gene polymorphism is the most studied biomarker for ADCC and several reports have already demonstrated its correlation with the efficacy of monoclonal antibodies treatment in solid tumors (39, 15, 40). One of the goals of this work was to define the impact of FCGR3A gene polymorphisms together with KIR/HLA-C interaction on BC pathogenesis and malignancy. While FCGR3A gene polymorphisms alone did not show a prominent role in the development of BC, the 48–158 haplotypes in the context of particular KIR genes had different correlations in BC patients compared to female controls. Our results suggested the possible existence of a cooperation between these genetic factors involved in ADCC response and KIR/HLA functional repertoire.