Triple negative breast cancer (TNBC) is a heterogenous disease often characterised by aggressive biology and poor prognosis. Efforts to precisely treat TNBC have been compounded by the lack of specific therapeutic molecular targets. Recent transcriptomic studies have revealed, among others, an immunomodulatory subtype of TNBC, whereby activated immune response genes are associated with good prognosis. Since then, a great deal of effort has been made to understand the immune microenvironment of some TNBC subtype, which comprises several immune cell populations including lymphocytes and macrophages. There is increasing evidence that the basal subtype may be significantly regulated by tumour-infiltrating T-cells and that high levels of tumour-infiltrating CD8+ T-cells may be a reflection of improved prognosis with chemotherapy sensitivity in TNBC. On the other hand, tumour-associated macrophages have been associated with a relatively poor outcome in TNBC. Comparison of the immune signatures in TNBC with non-TNBC may furthermore help us to understand these immune mechanisms potentially leading to new therapeutic approaches. Within this short review, we discuss the current scientific evidence regarding (a) the role of tumour-infiltrating lymphocytes in the clinical outcome in TNBC and (b) the newly discovered immunomodulatory genotype that may provide for a therapeutic target in TNBC.
- Breast cancer
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Triple negative breast cancer (TNBC) is defined by the absence oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) protein expression on immunohistochemistry. TNBC remains an important challenge in today's clinical practice because of the lack of therapeutic targets and poor clinical outcomes compared with non-TNBC.1 ,2 Research efforts to develop new effective targeted agents in TNBC have been hampered by a lack of a clear understanding of the underlying biology. Recent gene expression studies have identified tumour immune response genes as an important component in the pathobiology of TNBC.2 These findings have led to the development and use of immunomodulating agents in several ongoing TNBC clinical trials.3 ,4 In this review, we discuss the immune response gene signatures of TNBC both in a pathological and in a molecular aspect.
Clinicopathological characteristics of TNBC
TNBC represents 10%–17% of all breast carcinomas and is associated with young age, advanced disease at presentation, increased risk of visceral metastases, high histological grade and high density of lymphocytic infiltrate on histopathology.1 ,2 Histologically, TNBC mainly comprises invasive ductal carcinomas not otherwise specified. From a molecular perspective, Lehmann et al3 reported six distinct TNBC subtypes, including basal-like (BL1 and BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem–like (MSL) and luminal androgen receptor (LAR) subtype by gene expression analysis. BL1 and BL2 subtypes have higher expression of cell cycle and DNA damage response genes. The IM subtype is characterised by elevated immune response genes. M and MSL subtypes are enriched in gene expression for epithelial–mesenchymal transition (EMT) and growth factor pathways. LAR subtype is ER-negative, but exhibits enriched gene expression of steroid synthesis and androgen/oestrogen metabolism. Basal-like breast cancers are characterised by expression of high-molecular weight keratins (CK5/6, CK14 and CK17), vimentin, p-cadherin, fascin and caveolins 1 and 2.1 Basal-like breast cancers are associated with young patients, high histological grade, lymphocytic infiltration, lack of hormone receptor and HER2 expression, and have a worse prognosis than TNBC without basal marker expression.1 In this way, TNBC and basal-like breast cancers have a significant overlap and the rate of TNBC cases which express basal markers are quite significant (56%–84%).1 However, findings elucidating (a) IM subtype, enriched in immune cell markers, (b) increased expression of T lymphocyte-related genes, immune transcription factors and antigen processing, indicate that these would be biomarkers useful for patient selection in TNBC clinical trials as well as identification of potential markers of response to immunotherapy.3
Tumour-associated inflammatory cell infiltrate was initially thought to act as a catalytic agent or as a cocarcinogen aiding or expediting the development of neoplasia.4 Subsequent studies gave rise to an alternative view, whereby it was thought to reflect defence against cancer. Within the various histological categories of lung carcinoma, the quantum of inflammatory cellular infiltration was highest in squamous cell carcinomas and lowest in small cell carcinomas.5 There is strong evidence in some cancers that these infiltrating immune cells confer better prognosis, thereby supporting the theory of an effective antitumour immune response with inhibition of tumour progression and subsequent metastasis.4–7
Cancer and associated immune microenvironment
In 1949, a relationship between tumour-infiltrating lymphocytes (TILs) and prognosis was first reported in breast medullary carcinoma by Moore and Foote.6 Since then, a large number of studies on TILs have been published.5 ,7–11 The predominant component of TILs in solid tumours is CD3+ T-cells with CD20+ B-cells infiltrates seen infrequently. The population of CD3+ T-cells is further subclassified into CD8+ cytotoxic T-cells, CD4+ helper T-cells and CD4+ regulatory T-cells (Tregs). CD8+ T-cells are restricted to the recognition of antigen presented by major histocompatibility complex (MHC) class I molecules, whereas CD4+ helper T-cells recognise antigen presented by MHC class II molecules.7 Most tumour cells are positive for MHC class I, but negative for MHC class II, so the primary antitumour immune responses are preferentially dependent on CD8+ T-cells.7 The presence of a significant population of CD8+ TILs within a tumour is generally associated with favourable prognosis in a variety of cancers, including colorectal cancer, oesophageal cancer, pancreas cancer and non-small cell lung cancer.8–11 The CD4+ helper T-cell response is classified into Th1 and Th2 subtypes based on their cytokine profile, where Th1 preferentially elicits cellular immunity and Th2 elicits humoral immunity. Thus, the role of CD4+ helper T-cells in tumour immune response is to assist in activation of CD8+ T-cell mediated cell killing.7 The combination of high CD4+/high CD8+ TIL ratio has reported higher survival rate than other combinations, suggesting that CD4+ helper T-cells are required to elucidate a more effective response by CD8+ T-cells in non-small cell lung cancers.11 ,12 Tregs comprise 10% of total CD4+ T-cells in healthy peripheral blood and its population may be increased up to 30%–50% in tumour sites.13 Though Tregs play an important role in immune suppression and angiogenesis, in some tumour types, such as ovarian cancer,14 high levels of Tregs TILs is associated with worse prognosis. However, opposing findings have been observed in head and neck squamous cell carcinoma15 and urinary bladder cancer.16
Tumour-associated macrophages (TAMs) are composed of two distinct phenotypes: (a) type 1 macrophage (M1), which releases proinflammatory cytokines and induce Th1 immune responses, and (b) type 2 macrophage (M2), which inhibits Th1 immune responses by producing interleukin (IL)-10 and transforming growth factor β (TGF-β).17 A large number of studies have investigated the prognostic value of TAMs in variety of solid tumours and a significant proportion of them have reported that high TAMs infiltration levels are correlated with poor prognosis.18 This protumour effect of TAMs appears to be mediated through tumour proliferation, angiogenesis, matrix remodelling and production of significant growth promoting and sustaining cytokines, such as epithelial growth factor, IL-6, vascular endothelial growth factor and matrix metalloproteinases.17
Inflammatory infiltrates in TNBC
There are divergent opinions on the relationship between CD8+ TILs and prognosis in breast cancer. Recently, Mahmoud et al observed that neither intratumorous CD8+ TILs nor adjacent stromal CD8+ TILs are significantly associated with patient outcome. However, high total (intratumorous, adjacent stromal and distant stroma) number of CD8+ TILs and distant stromal CD8+ TILs was associated with better prognosis.19 Liu et al20 have shown that presence of intratumorous, stromal and total CD8+ TILs is associated with better survival only in basal-like breast cancers, but not in non-basal TNBC or in other intrinsic molecular subtypes. On the same note, Baker et al21 demonstrated significant association between the presence of CD8+ TILs and better prognosis in ER-negative and high-grade breast cancers, whereas this significant correlation of lymphoid infiltrate with prognosis was not observed when all breast cancer subtypes were analysed together. In addition, gene expression profiling has shown an association between overexpression of immune response gene and better prognosis in ER-negative breast cancers.22
Studies elucidating the role of CD4+ helper T-cells in breast cancers are few in number. A small study, which refers to immune response pathway via Th1 and Th2, proposed that in ER-negative breast cancers (a) increased IL-2 and interferon γ production which are induced by Th1 activation are correlated with good prognosis, whereas (b) increased IL-13 and TGF-β, which are raised by Th2 activation, are associated with poor prognosis.23
Though there is fairly general agreement that high levels of Tregs TILs are correlated with high histological grade and ER negativity,24–27 opinions are divided among researchers on the relationship between Tregs TILs and clinical outcome. Mahmoud et al25 has shown the quantum of Treg is not an independent prognostic factor in their study of 1445 cases of invasive breast carcinoma. In contrast, Bates et al27 have revealed that there is a significant correlation between the high number of Tregs TILs and poor prognosis in ER-positive breast cancers. Others have reported the association of high levels of Tregs TILs with good prognosis in ER-negative breast cancers28 and in TNBC29 (see table 1).
There are reports that increased number of TAMs is correlated with high vascular density and hence poor outcome in breast cancers.30 However, a large study involving 1322 patients has reported that TAMs are not an independent prognostic marker in breast cancers.31 Similarly, Medrek et al32 have reported no significant difference in outcome based on TAMs in TNBC, but intratumorous CD163+ macrophages were positively correlated with high histological grade, larger size, Ki-67 positivity, hormone receptor negativity and TNBC. TAMs in basal-like breast cancers have been reported to show difference in macrophage polarisation, cytokine profile and migratory function compared with TAMs in luminal cancer,33 suggesting that immune response mediated by TAMs in basal-like breast cancer may be different from that of non-basal breast cancer. The reason why some TNBC tumours have a propensity to incite an inflammatory reaction is not well known, although some plausible hypotheses have been proposed. Recently, increased expression of lactoferrin has been identified in TNBC.34 Lactoferrin has been shown to reduce expression of ER, PR and HER2 and increase expression of endothelin-1, suggesting that lactoferrin contributes to the development and invasiveness of TNBC.35 Although the role of lactoferrin as a proinflammatory or anti-inflammatory factor is still unresolved, some studies have shown lactoferrin-enhanced antitumour immune response and production of proinflammatory cytokines derived from macrophages and dendritic cells.36 ,37 Taken together, it is possible that lactoferrin influences TNBC tumourigenesis and tumour immune response.
EMT and immune response in cancer
EMT, defined by loss of epithelial characteristics and the acquisition of a mesenchymal phenotype, is regarded as an important biological process involved in tumour invasion and metastasis.38 ,39 Recent findings have hypothesised that TILs promote EMT and acquisition of breast cancer stem cells (BCSC) properties40 and that these phenomena are preferentially observed in breast cancers with basal-like phenotype.41 ,42 There is evidence to suggest that immune effectors can induce EMT following an acute or chronic inflammatory response to tumour infiltrate. Although the exact mechanism is unknown, it is possible that CD8+ T-cells present in the tumour microenvironment can produce direct mediators of EMT.40
Impact of chemotherapy in immune microenvironment of TNBC
Chemotherapy remains the mainstay for treatment of patients with TNBC. Thus, clinicopathological predictors for chemotherapy response are very important. Recently, some studies have revealed that high levels of CD8+ TILs in cancer cell nests heighten neoadjuvant chemotherapy sensitivity in breast cancers,43–46 ER-negative breast cancers47 and TNBC.48 Interestingly, in one study, 7 out of 21 TIL-negative cases showed conversion into TIL-positive cases after chemotherapy and converted TIL-positive cases were seen in patients with clinical partial or complete response and not seen in patients with stable disease.49 This observation led to the suggestion that chemotherapy induces cytotoxic effect and influences tumour immune response in this setting. The underlying mechanisms are yet unclear, but three processes have been proposed: (a) dying tumour cells induced by chemotherapy mediate induction of antitumour T-cells. High-mobility group box 1 released by dying tumour cells in the late phases of apoptosis promotes tumour antigen presentation by dendritic cell via activation of Toll-like receptor 4 and antigen-presenting cells induce immune cells activation, (b) chemotherapy decreases immunosuppressive Tregs while the number of CD8+ T-cells remaining unaffected and (c) chemotherapy sensitises tumour cells to CD8+ T-cells acting supported by TNF-related apoptosis-inducing ligand.50–52
Tumour-associated antigens of TNBC
Recognition of tumour antigen is required for activation of CD8+ T-cells, as previously mentioned. Tumour-associated antigens are classified into several groups based on their expression pattern: (a) products of abnormally activated genes that are not expressed in normal somatic tissue as represented by cancertestis (CT) antigen, for example NY-ESO-1, (b) differentiation antigen that is specific for the tissue of origin, for example NY-BR-1, (c) ‘mutation’ antigen that is produced by aberrant genes, for example p53 and (d) viral antigen that is derived from product of oncogenic virus function, for example human papilloma virus proteins.4 ,53 Due to restricted pattern of expression, CT antigens often have been object of study related to immunotherapy-based treatment strategies. Grigoriadis et al54 found that CT antigen expression was frequently seen in ER-negative breast cancers. Subsequently, a few studies supporting this theory have revealed that CT antigens such as MAGE-A and NY-ESO-1 are preferentially expressed in TNBC or hormone receptor negative and high histological grade breast cancers.54 ,55 Immunohistochemically, frequency of CT antigen expression is 12%–26% in ER-negative breast cancers versus 2%–6% in ER-positive breast cancers.54–56 Ademuyiwa et al56 have shown that NY-ESO-1 expression is correlated with high levels of CD8+ TILs in TNBC, while Karn et al57 showed that poor prognosis of patients with high MAGE-A expression is ameliorated by the concomitant upregulation of immune cell metagenes in TNBC.
Immunosuppressive factors in TNBC
In addition to antitumour immune response, tumour evasion of immune surveillance is another important aspect that takes place via a combination of mechanisms involving immune checkpoints, tumour-mediated immune tolerance and cytokine-induced immune suppression. In immune checkpoint mediated immune suppression, T-cell costimulation and coinhibition signals derived from interaction between T-cells and antigen presenting cells play a central role in regulation of the adaptive immune response. T-lymphocyte antigen 4 (CTLA-4) receptor as well as the PD-L1 ligand and PD-1 receptor are well established as coinhibitory partners. CTLA-4 has attracted attention as immunotherapy target, but studies have been limited to mainly in vitro studies.57 PD-L1 is constitutively expressed on all haematopoietic cells and most non-haematopoietic cells, whereas PD-1 is expressed on activated T-cells and B-cells and myeloid cells. The interaction of these two molecules induces inhibition of CD4+ and CD8+ T-cell proliferation by arresting the T-cell cycle, thereby mediating immune tolerance.58 In breast cancers, high levels of PD-L1 expression are associated with histological grade 3, ER-negative, PR-negative and high Ki-67 index.59 ,60 In vitro studies with breast cancer cell lines show that a subset of basal-type breast cancer cell lines express higher levels of PD-L1 compared with other basal and luminal breast cancer cell lines. These high expressing PD-L1 basal cells differentially express genes involved in motility, invasion and drug resistance at a higher level compared with basal cell lines expressing low PDL1.61 Early clinical results using inhibitory agents against the PD-1 pathway have demonstrated its importance in maintaining an immunosuppressive tumour microenvironment and by acting as a target for cancer immunotherapy.62 It is likely that PD-L1/PD-1 signalling also influences immune response of high-risk breast cancer, such as TNBC as seen from studies where PD-L1-positive tumours had greater CD8+ T-cell infiltrate than tumours lacking PD-L1.63
Dendritic cells (DCs) are one of the tumour-infiltrating immune cells and play as important regulators of host immune system. Based on expression markers, DCs are classified into two main subtypes: myeloid DCs and plasmacytoid DCs. Tumour-infiltrating DCs that express S-100 and CD1a are not correlated with prognosis in breast cancers.64–66 However, a recent study has identified that tumour-associated plasmacytoid DCs may preferentially infiltrate TNBC compared with non-TNBC and contribute to immune tolerance through repression of interferonα and Tregs expansion.67 Cytokines are produced by immune cells and by cancer cells. In breast cancers, ER-negative breast cancers have been shown to express high levels of cytokines.67 IL-6 production is upregulated especially in TNBC, which in turn induces colony formation as well as cancer cell proliferation in cancer cell lines.68 ,69 Supporting its role in tumourigenesis, IL-6 reduction induces enhanced CD4+ and CD8+ T-cells response in vitro.70 Altogether, it seems reasonable to hypothesise that IL-6 suppresses antitumour immune response in TNBC and that IL-6 inhibition may provide a therapeutic strategy against TNBC.
Gene signatures in ER-negative breast cancer
Gene profiling of immune markers has been used to subdivide TNBC into distinct entities.71–73 For instance, a high B-cells and low IL-8 metagenes have been associated with good prognosis in this population, suggesting that IL-8 suppression may be a therapeutic approach.74 In another study, a group of 14 genes linked to immune/inflammatory cytokine regulation identified TNBC patients with a good outcome in the absence of aggressive adjuvant therapy.75 The outcome of these studies taken together, suggest that in ER-negative breast cancers, especially TNBC, the clinical status is particularly influenced by tumour-related immune responses. More recently, the basal endoplasmic reticulum stress has been found to be activated in TNBC, in turn cooperating with hypoxia response signalling, thereby promoting tumour progression and early relapse.76 ,77 Interestingly, the hypoxia response pathway mediated by hypoxia-inducible factors has been found to influence the cancer immune response through modulation of T-cells and myeloid cells differentiation and function.78
Recent evidence clearly demonstrates a relationship between TILs and clinical outcome; high levels of CD8+ TILs indicate a good prognosis in TNBC. Further investigations evaluating the role of Treg TILs in breast cancer and its relationship with CD8+ cells and clinical outcomes are required. In addition, teasing out the relative contribution of the other immune cells in the tumour microenvironment, such as TAMs, and cytokines production will improve our understanding of the biology of TNBC and help inform treatment strategies and therapeutic drug development.
Take home message
Recent transcriptomic studies have revealed an immunomodulatory subtype of triple negative breast cancer (TNBC) whereby activated immune response genes are associated with good prognosis.
High levels of tumour-infiltrating CD8+ T-cells may be associated with improved prognosis with chemotherapy sensitivity in TNBC.
Tumour-associated macrophages have been associated with a relatively poor outcome in TNBC.
Hypoxia response pathway mediated by hypoxia-inducible factors (HIFs) influences the cancer immune response by modulation of T-cells and myeloid cells differentiation and function.
Dissecting the relative contribution of the immune cells in the tumour microenvironment will improve the understanding of TNBC biology and influence treatment strategies and development of targeted drugs.
Handling editor Cheok Soon Lee
Contributors JI and HM: Contributed the idea and drafted the manuscript. RD, PHT and S-lK: Critically edited the manuscript.
Funding This study was funded by the grant SMPO201302.
Competing interests None.
Provenance and peer review Not commissioned; internally peer reviewed.
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