The composition of T cell infiltrates varies in primary invasive breast cancer of different molecular subtypes as well as according to tumor size and nodal status

In tumor microenvironment, lymphocytes predominate in mononuclear infiltrates and represent an adaptive antitumor immune response. The most abundant population of tumor-associated lymphocytes is T cells, which include many subpopulations differing in their function. Among them, CD8+ cytotoxic T lymphocytes (CTLs) are particularly known for their cytolytic activity against cancer cells. On the contrary, GATA3+ T helper 2 (Th2) cells and FOXP3+ regulatory T lymphocytes (Tregs) downregulate antitumor immune response by impairing antigen presentation, activity, and cytotoxicity of other immune cells, thus promoting tumor growth and immune tolerance. Tregs originate from naïve T cells both in the thymus and at the periphery, and the process of their differentiation is orchestrated by a specific cytokine milieu. Molecules secreted by tumor-infiltrating lymphocytes (TILs), cancer cells, and other components of tumor microenvironment affect the composition and function of the cancer milieu, and, thereby, modulate the course of breast cancer progression [1‐6]. It was observed that TILs rich in Tregs interact with cancer-associated fibroblasts, contributing to stromal remodeling, that presumably promote tumor growth and invasion [7]. The density of T cells was reported to increase as mammary tumor progresses from normal breast tissue, through benign and in situ lesions, to invasive ductal cancers; this finding was interpreted as a stepwise increase in immunity with the course of mammary tumorigenesis [5‐8]. An antitumoral immunity shows plasticity (immunoediting) and changes in time from tumor elimination (based highly on CTLs), through the equilibrium phase to immune escape (characterized by immunosuppressive profile of tumor microenvironment and generation of Tregs). The process of immunoediting is thought to result from the shifted balance between respective T cell phenotypes and selection of non-immunogenic clones [9, 10]. In line with these, the immune response appears to be dysfunctional and skewed toward suppression in invasive breast tumor tissue [2, 11].

Since the discovery of the intrinsic molecular subtypes that differ in their genetic pattern and clinical aggressiveness, invasive breast cancer has become regarded as a heterogeneous disease [12, 13]. To a certain extent, the interplay between malignant breast tumor and TILs is dependent on tumor genetics and biology [10]. Moreover, there is growing evidence that prognostic and predictive relevance of TILs varies in breast cancer of different intrinsic subtypes [1, 4, 5, 10, 11]. Nonetheless, the relationships between composition of lymphocytic milieus and breast cancer molecular subtypes have not been fully elucidated so far. Relationships between cancer and its microenvironment are of great interest, as some chemotherapeutic agents may elicit or enhance antitumor immune reactions, and innate, adaptive, cellular, and humoral pathways may be involved in cancer cell killing [14]. Simultaneously, new therapeutic approaches that aim at inducing potent immune response are sought. This includes an increase of tumor immunogenicity, inhibition of immune evasion [10, 11], and enhancement of cytotoxic and Th1 response, as well as a reduction of regulatory and Th2 cell impact on neoplastic breast tissue [5].

In our study, we investigated the lymphocyte infiltrate composition in order to assess its relationships with invasive breast cancer molecular subtypes and the occurrence of other prognostic and predictive markers for this disease. For this purpose, we evaluated both numbers of CTLs, Tregs, and Th2 cells and their percentages in tumor-associated immune infiltrates. Moreover, we also calculated proportions of investigated cells to assess differences in their relative quantities with regard to clinico-pathological indicators in breast cancer.

A detailed description of the study group is shown in Table 2.

CTLs are commonly considered as a part of cancer immune surveillance. Some research into breast cancer-linked CTLs revealed their lytic and proapoptotic activity [18] as well as a memory phenotype in the majority of this cell population, particularly when high-grade lesions were concerned [19]. Tsang et al. [20] observed that CD8+ and FOXP3+ expressions were mutually exclusive in double immunohistochemical staining of breast cancer microenvironment. On the contrary, the existence of fractions of CTLs that express FOXP3 secrete immunosuppressive interleukin (IL)-10 [21] and co-express molecules associated with anergy, exhaustion, or unresponsiveness in tumor-infiltrating lymphocytes [22] was reported.

The data on associations between lymphocytic infiltrate and breast cancer molecular subtype are inconclusive. In line with our study, some authors reported that increased CTL counts were associated with ER and PR negativity, HER2 overexpression [23‐25], and higher Ki67 level [25, 26] in breast cancer, while the results from other publications question these findings [20, 26, 27]. Liu et al. hypothesized that the location of immune infiltrates (intraepithelial or peritumoral) may influence activation of its cells, as cell populations within a tumor are dispersed and their interactions are impeded [23]. In the Miyan et al. study, a significantly increased number of CTLs was observed at the invasive margin of basal-like and luminal B/HER2+, with their lowest counts in luminal A lesions [25]; however, the authors applied St Gallen 2013 molecular subtype classification in their research. Tsang et al. suggested that the mechanism of intratumoral recruitment and survival of lymphocytes may differ between subtypes, and that in HER2-positive tumors, CTL migration is preferred over Treg influx [20]. Our observation of high CTL counts in HER2+ non-luminal and triple-negative phenotypes was made for a population of these cells located at tumor edge, exclusively. On the other hand, the percentages of CTLs in tumor-associated infiltrates at invasive margin did not differ between subtypes. Therefore, the increased numbers of CTLs may reflect more abundant TILs noted in these breast cancer subtypes rather than a shift in immune response toward more potent cell killing. A favorable prognostic value of high levels of breast cancer-related CTLs was attributed to ER-negative as well as ER+/HER2+ phenotypes [28]. Moreover, high FAS protein expression in ER-negative cancers was proposed to be one of the contributing factors to beneficial impact of CTL on patient survival. Of note, their adverse effect on ER-positive FAS-high patient outcome indicated different functions of tumor-infiltrating CTLs with respect to breast cancer subtypes [29].

Literature data on relationships between CTLs and other prognostic factors in invasive breast cancer are also ambiguous. Some authors [14, 19, 23‐25], but not all [23, 27, 30], report higher CTL counts in tumors of higher histological grade and size, which is in line with our findings. We noted that relationships between more numerous CTL infiltrates and higher grades concerned both intraepithelial area and tumor edge, while tumors of greater diameter were characterized only by a more abundant intraepithelial population. After stratification, the latter finding remained significant for luminal lesions, exclusively. Regarding lymph node metastases, higher counts [26, 27, 31] as well as a higher frequency of CTLs [19] were observed in primary tumors with nodal spread, but such association was not observed by other groups [23, 32]. In our study, the CTL infiltrates did not differ according to nodal status.

FOXP3 is a transcription marker expressed in a vast majority of breast cancer-infiltrating Tregs [33]. Similar to CTLs, Tregs were observed to accumulate in breast tumor and its immediate milieu, in comparison with normal tissue. Moreover, tumor-infiltrating Tregs were more frequently characterized by activated, strongly immunosuppressive but exhausted phenotype, which may correspond with immune tolerance [22]. As far as the breast cancer intrinsic subtypes were concerned, the most numerous regulatory T cell infiltrate is frequently associated with either TNBC or HER2+ non-luminal phenotype of tumors, while the lowest Treg numbers are observed in luminal A lesions [23, 25, 34], which is in accordance with our results. Moreover, the stronger Treg infiltrate of TNBC concerns both the surrounding stroma and tumor center. Such findings indicated associations between tumor biological features and immunological response in invasive breast cancers [25] as well as more immunosuppressive microenvironment of clinically aggressive subtypes [23]. Moreover, the increased proportion of Tregs in a lymphocytic milieu [35], as well as their higher quantities, was noted in TNBC and hormone receptor (HR)-negative and HER2-overexpressed breast tumors [23, 34], which supports our results. We also observed slight correlations between Treg percentage in immune cell infiltrates of invasive edge and either a drop of PR or an increase in Ki67 expression that suggest a shift toward a more immunotolerant milieu in PR-negative or intensively proliferating breast cancers. More abundant Tregs were found in the center of ER-positive and in the peritumoral area of ER−/HER2+ cancers, while a lower number of cells infiltrated the intratumoral site of ER−/HER2+ tumors in Tsang et al. research [20]. Some authors did not observe any differences in Treg infiltrates regarding breast cancer intrinsic subtypes [36]. In ER-negative tumors, more intensive Treg infiltration was associated with better disease-free survival [33]. Similar to CTLs, higher levels of Tregs were often observed in high-grade cancers [14, 23, 35], but their relationship with tumor size and nodal status is controversial [22, 23, 26, 34]. We found increased Treg numbers and percentages in luminal breast tumors of greater size, as well as a higher proportion of Tregs in the microenvironment of node-positive invasive tumors, that suggests a regulatory bias in TILs of more advanced cancers.

The ratio of CTL to Treg numbers is regarded as an indicator of cytotoxicity. A higher CD8+/FOXP3+ ratio was observed by Liu et al. in the peritumoral area of non-luminal breast cancers and indicated greater cytolytic potential of the lymphocytic milieu surrounding these tumors [23]. It was postulated that the change of this parameter is rather due to Treg reduction than CTL recruitment [14]. Complementary FOXP3+/CD8+ cell ratio was suggested to reflect an immune evasion of a tumor, with its higher values in tumor center as compared with the peritumoral area. In breast tumor, it was associated with ER negativity, higher proliferation rate, and high histological grade, but not with tumor size or nodal involvement [25]. In our study, increased intraepithelial CTL/Treg number ratio was associated with lobular histology and metastatic disease of non-luminal cancers. Thus, we hypothesize that, for breast tumors of non-luminal phenotype, their spread is associated with cytotoxicity failure.

Information on tumor-associated Th2 cells in breast cancer is scarce. Th2 was reported as a predominant population of T helper cells in a mouse model of luminal breast cancer; their lower counts in the tumor milieu were associated with decreased pulmonary metastasis by Zhang et al. [37]. Moreover, higher levels of Th2 cytokines—IL-10 [14] and IL-5 [3]—were related to the lack of pathologic complete response after chemotherapy and worse survival in breast cancer patients, respectively. In our study, tumor-associated Th2 cells showed a slight correlation with HR. On the contrary, the expression of genes related to Th2 signaling was more prominent in basal cancers by Kristensen et al. [38]. Ghirelli et al. showed that cytokines secreted by breast cancer tissue resulted in regulatory Th2 bias of tumor-related immunity, which supposedly was GATA3 independent [39]. To assess immunoregulatory and suppressive potential of immune infiltrates, we evaluated the Treg/Th2 cell percentage ratio. Its high value was associated with adverse prognostic indicators: HER2+ non-luminal subtype, decrease in HR expression, increasing proliferation rate, and higher grade as well as greater tumor size and positive lymph node status, particularly in luminal cancers. In addition, the higher percentage ratio of CTLs to Th2 at invasion front modestly correlated with HR expression. Thus, we hypothesize that the bias of immunosuppressive microenvironment toward regulatory function is associated with clinically more aggressive breast cancers.

Our recent research studies aimed at evaluation of tumor microenvironment in primary invasive breast cancer have shown associations between higher quantities of mast cells and beneficial prognostic indicators [40] and relationships between T cell, B cell, and NK cell infiltrates and adverse clinical factors [41]. These findings induced us to presently investigate infiltrates of several T cell subpopulations in this disease. In conclusion, we observed that T cell infiltrates of primary invasive breast tumors differ in numbers and percentages of its individual populations regarding cancer molecular features and prognostic markers. Moreover, the relationships between lymphocytic composition and pT or nodal status vary according to cancer phenotype, suggesting that distinct mechanisms govern cancer progression in luminal and non-luminal lesions. As more numerous T cell subpopulations in breast cancer tissue may result from higher TIL levels, we additionally evaluated percentages of analyzed cells in tumor-surrounding infiltrates that should be resistant to the number of TILs. Thus, further investigation is needed to elucidate function of immune infiltrates in breast cancers of different molecular subtypes.