The immune system is active in breast cancer, playing a dual role in tumor progression and in immune surveillance. Infiltrating immune cells are both prognostic and predictive of response to standard breast cancer therapies. Breast cancer vaccines can activate and expand tumor-specific T cells, but have enjoyed minimal clinical success to date. Immune checkpoint blockade is a new approach to cancer immunotherapy, with documented clinical responses in diverse tumor types. Interest in breast cancer immunotherapy has been reignited by recent reports of objective responses in metastatic triple-negative breast cancer with both pembrolizumab (a programmed cell death protein 1 [PD-1] antagonist) and MPDL3280A (a programmed cell death ligand 1 [PD-L1] antagonist). Rational strategies for combination immunotherapy that expand and promote the trafficking of tumor-specific T cells, support their activity at the tumor site, and abrogate pathways of immune suppression within breast tumors are most likely to result in objective responses that translate into long-term disease control and cure.
The immune system plays an integral and complex role in breast cancer biology, both promoting tumor growth and mediating the eradication of disease. Understanding this seemingly contradictory role requires insight into the dynamic interplay between various immune effector cells, tumor cells, stromal cells, and soluble factors; the theory of cancer immunoediting provides context for these considerations. According to this hypothesis, tumor variants capable of surviving cancer immune surveillance are selected through genetic evolution, while those that cannot survive tumor surveillance are eradicated. The interaction between the immune system and tumors occurs in three phases: elimination, equilibrium, and escape. In the elimination phase, an acute inflammatory response triggered by stromal remodeling and angiogenesis initiates recruitment of innate immune cells (macrophages, dendritic cells, natural killer cells, and other cells) into the tumor microenvironment. Recognition of transformed tumor cells by these cells results in the production of proinflammatory cytokines, most notably interleukin-12 (IL-12) and interferon-γ (IFN-γ). These cytokines promote further activation of innate immune cells and tumor cell death. Also during this phase, dendritic cells mature, process tumor-associated antigens, and migrate into tumor-draining lymph nodes, where they present antigen to activate naive, tumor antigen–specific CD4+ and CD8+ T cells. These activated T cells expand and home to the tumor microenvironment, where they facilitate tumor cell death. The ultimate fate of the tumor can proceed in two directions: the complete eradication of tumor, or the evolution of tumor cell variants that escape the immune response and establish measurable tumors. Selective immune pressure on tumor cells can result in the accumulation of tumor cells with defects that predispose them to escape immune surveillance. These include loss of major histocompatibility complex (MHC) class I protein expression, other defects in antigen processing and presentation pathways, defects in T-cell receptor (TCR) signaling and costimulation, mutation or loss of tumor antigens, and deficiencies in IFN signaling pathways.[2-3] During the equilibrium phase, inflammation shifts from acute to chronic, ultimately leading to complete tumor escape from immune surveillance and to tumor outgrowth. Newly recruited tumor-associated cells (macrophages, regulatory T cells [Tregs], myeloid-derived suppressor cells, activated B lymphocytes, and tumor-associated fibroblasts) propagate chronic inflammation and tumor progression. Major mechanisms that actively promote tumor growth include the inhibition of tumor antigen–specific T cells by intratumoral regulatory T cells, a shift from an antitumorigenic T helper type 1 (TH1) immune response to a protumorigenic T helper type 2 (TH2) immune response, and the production of soluble factors by tumor cells that directly inhibit dendritic and T-cell function while promoting angiogenesis and stromal remodeling.[2,4,5] Furthermore, the upregulation of inhibitory immune checkpoint pathways—like the programmed cell death protein 1 (PD-1) pathway—by tumor cells and immune cells within the tumor microenvironment further inhibits the activation of tumor antigen–specific T cells.[5,6] Ultimately, tumors acquire complete autonomy from immune surveillance and grow and metastasize unchecked. This is the escape phase. Understanding the interplay of these cells and regulatory pathways in breast cancer will provide the blueprint for effective breast cancer immunotherapy, reversing the balance to promote tumor rejection by the immune system.
Immune Biomarkers in Breast Cancer
The impact of the immune milieu on breast cancer progression and outcomes depends upon both the carcinoma phenotype and inflammatory cell subsets within the breast tumor microenvironment (Figure 1).[2,7] For instance, it has long been recognized that medullary breast carcinomas carry a favorable prognosis, and are characterized by syncytial high-grade carcinoma cells with a prominent lymphoplasmacytic infiltrate. Breast carcinomas can be further subdivided by gene expression profiles into intrinsic molecular subtypes[9-12] that have immunohistochemical surrogates based on tumor expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2).[13,14] These subtypes include luminal A (ER+PR+HER2−Ki67low), luminal B (ER+PR+HER2−Ki67high or ER+PR+HER2+), HER2+ (ER−PR−HER2+), and basal-like (typically triple-negative [ER−PR−HER2−] disease with expression of cytokeratin 5/6 and/or epidermal growth factor receptor).
Triple-negative and HER2+ breast carcinomas are thought to be more immunogenic than luminal A carcinomas, as evidenced by the tumor-infiltrating lymphocyte (TIL) composition within the tumor microenvironment, as well as carcinoma gene signature patterns. Higher numbers of TILs are seen in ER– carcinomas compared with ER+ carcinomas. Immune response gene expression modules are associated with better survival in ER−HER2− and HER2+ carcinomas, but not ER+HER2− carcinomas. A T-cell metagene profile (lymphocyte-specific kinase metagene) correlated with improved response to chemotherapy in all ER− carcinomas and ER+HER2+ carcinomas. B-cell gene profiles conferred a favorable prognosis in triple-negative breast carcinomas, ER− and ER+Ki67high carcinomas, but not ER+Ki67low carcinomas.[17-19] In contrast to luminal carcinomas, basal-like carcinomas had high expression of nonfavorable TH2/protumor humoral immunity genes.
Tumor Infiltrating Lymphocytes and Lymphoid Aggregates in Breast Cancer
Association with survival
Both TILs and lymphoid aggregates have been associated with survival in breast cancer. The presence of TILs within the breast tumor microenvironment can be assessed by hematoxylin and eosin–stained sections, and immunohistochemistry (IHC) can be used to assess lymphocyte subsets and specifically characterize the TIL phenotype. The presence of TILs in treatment-naive triple-negative breast carcinoma is an independent prognostic factor for improved overall survival,[22,23] decreased distant recurrence,[23,24] and increased metastasis-free survival. In addition, the presence of TILs in residual triple-negative breast carcinoma after neoadjuvant chemotherapy is also prognostic for metastasis-free and overall survival. The presence of brisk TILs in treatment-naive HER2+ carcinomas correlates with prolonged survival and response to trastuzumab. Among specific TIL subsets, high numbers of CD8+ cytotoxic T cells, CD4+ follicular helper T cells, and CD20+ B cells are predictors for patient survival across breast carcinoma subtypes. Conversely, high numbers of forkhead box–binding protein–3 (FoxP3)+ Tregs are associated with higher tumor grade,[29,30] ER negativity, shorter relapse-free survival,[29,31] and shorter overall survival time. Consistent with these findings, metastatic triple-negative breast carcinomas at first relapse have fewer TILs than their matched primary breast carcinomas. The clinical significance of tumor-infiltrating macrophages remains less clear.
Lymphoid aggregates are another emerging biomarker for the inflamed tumor microenvironment. Also termed ectopic lymphoid-like structures or tertiary lymphoid structures,[7,21,35] these aggregates can vary from loose clusters of T and B lymphocytes to well-organized nodules of lymphocytes with germinal centers. These structures represent foci of immunoregulation that reflect an ongoing adaptive immune response. Similar structures have been observed in treatment-naive breast carcinomas, where the density of high endothelial venules (thought to be a gateway for TIL entry into tumors) is associated with a low risk of relapse and longer overall survival time.
Association with response to therapy
The presence of TILs and/or lymphoid aggregates in the breast tumor microenvironment can also predict response to neoadjuvant and adjuvant therapy. High numbers of TILs correlate with pathologic complete response (pCR) to neoadjuvant chemotherapy across breast carcinoma subtypes.[38-40] The presence of > 60% TILs or an immune subtype (based upon high mRNA expression of immune genes) correlates with pCR to neoadjuvant anthra-cyline-plus-taxane chemotherapy in triple-negative breast carcinomas and HER2+ carcinomas, particularly in patients treated with chemotherapy plus carboplatin. High levels of TIL infiltrates in ER− carcinomas predict pCR after neoadjuvant anthracycline-based chemotherapy, but not after treatment with cyclophosphamide, methotrexate, and fluorouracil (CMF). Greater numbers of TILs are also associated with decreased recurrence rates after treatment with adjuvant trastuzumab in HER2+ carcinomas. In addition, a greater reduction in FoxP3+ Tregs is seen in patients responding to the aromatase inhibitor letrozole, and the development of new TILs is associated with response to neoadjuvant paclitaxel.
Furthermore, the presence of TILs in the residual breast tumor microenvironment after neoadjuvant therapy is a favorable prognostic factor. Decreased levels of FoxP3+ Tregs are seen in patients with pCR.[26,45] The presence of high CD8+ and low FoxP3+ T-lymphocyte levels in residual tumors after neoadjuvant chemotherapy is associated with improved recurrence-free and overall survival; notably, a score combining the CD8/FoxP3 ratio and pathologic stage identifies a subgroup with an overall survival of 100%. The presence of T-bet+ lymphocytes within tertiary lymphoid nodules in residual HER2+ tumors after neoadjuvant therapy with trastuzumab and a taxane (therapies that promote a TH1 antitumor response) is also associated with improved recurrence-free survival.
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