The role of stromal immune microenvironment in the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer

CD20 is mainly expressed on B cells and in a subpopulation of T lymphocytes and follicular dendritic cells. B lymphocytes are CD20 + adaptive immune cells, which trigger humoral immunity through the production and secretion of antibodies which recognize specific tumor’s antigens and inhibit the functionality of the receptor. Additionally, antibodies are able to signal other cancer killing cells to eliminate tumor cell population [11]. In retrospective studies, B cells have been associated with favorable prognosis in invasive breast cancer [18]. Though this supports an anti-tumor role for B cells and antibodies, other studies have associated them with poor prognostic factors in breast cancer because human breast cancer cells can induce a regulatory phenotype in B cells, initiating the production of the transforming growth factor beta (TGF-beta), a cytokine that stimulates CD4 + T cells to become immunosuppressive T regulatory cells [19].

CD163 is expressed by tissue macrophages and monocytes. Tumor-associated macrophages (TAM) and their infiltration accompany a worse prognosis in ER- and ER + breast tumors [20]. TAM exerts pro-tumor effects: stimulate angiogenesis, metastasis, suppression of adaptive immunity and production of matrix proteins. The highest risk of DCIS recurrence is observed in patients with elevated M2 macrophages, suggesting that M2 macrophages may abrogate the tumor-fighting T cell function [7, 11].

In the present study, we did not find any relationship between CD4+, CD8+ and CD 138+ in primary DCIS and invasive breast cancer recurrence in the same patients. Similarly, we did not observe statistically significant differences between the scores of FOXP3 + immune cells in both groups; however, increase in the number of those cells after invasive progression was revealed. It was very difficult to analyze the role of FOXP3 + regulatory lymphocytes in the progression of DCIS because the score of these cells in the stroma in both groups was very low.

CD8 is found on a T cell subset of normal cytotoxic/suppressor cells and natural killer cells. CD8 + cytotoxic T lymphocytes are typically associated with anti-tumor immunity, although their activity can be suppressed in the tumor microenvironment [21]. They are important immune prognostic markers for the outcome of TNBC and HER2-positive invasive breast cancer and correlate positively with improved survival [22].

CD4 is expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells. There are a number of subsets of CD4 + T cells that have different functions within the tumor. T helper 1 (TH1) cells represent a subset of the CD4 + T cell population that typically expresses high levels of interferon gamma, which acts to limit tumor growth, promote antigen presentation and active macrophages M1. Contrary to TH1, the helper cells type 2 (CD4 + TH2) secrete cytokines which inhibit T-cell mediated cytotoxicity, stimulate macrophages and promote a pro-tumorigenic environment [4, 11, 21].

FOXP3 is specifically expressed in T regulatory cells (Tregs), including CD4⁺ CD25^highTreg cells and CD8⁺ CD25^high Treg cells. T regulatory lymphocytes are both CD4 + and FOXP3 + TH2 and have immunosuppressive functions. Normally, they help to protect against autoimmunity. In breast cancer, they are associated with poor prognosis as they contribute to the pro-tumor immune response and assist the tumor in subsequent immune escape. T regs CD4 + FOXP3 + allow the progression of the tumor by expressing inhibitory factors that inhibit the anti-tumor TH1 response [21, 23]. Thus, CD4 + Th1 cells, CD4 + CTLs exert anti-tumor activity, whereas regulatory T cells, CD4 + TH2 cells show tumor-promoting activity [4, 21, 23].

Our results concerning CD4+, CD8 + and FOXP3 + cells differ from the findings reported in other studies. Kim et al. [4] showed, basing on 590 cases, that the immune microenvironment of DCIS is different from that of invasive cancer. CD4+, CD8 + and FOXP3 + immune cell infiltration was significantly higher in invasive breast cancer compared to pure DCIS (p < 0.001). The comparison of the dominance of CD4 + versus CD8 + cells in pure DCIS and invasive cancer revealed that the infiltration of CD4 + TILs was higher than of CD8 + in pure DCIS (p < 0.001), while the reverse was true in invasive breast cancer with the CD8 + TILs being the dominant subset (p = 0.006) [4]. This is consistent with other studies [24, 25], but we did not observe such a relationship in our study. The difficulty in drawing a conclusion as to the role of CD4 + in our analysis is that CD4 + TILs display a large degree of plasticity and the ability to differentiate into multiple subsets in response to microenvironmental cues. Actually, we did not know whether the detected CD4 + cells in the present study acted in favor of (TH1 subset) or against (TH2 subset) tumor growth [4]. Thus, in further studies, analyses of CD4+ subsets would be crucial in establishing their role in tumor progression.

In our study, a comparison was made of 30 DCIS and 30 recurred invasive breast cancers in the same patients. To our knowledge, the only article concerning the comparison of the same patients with primary DCIS and its ipsilateral breast recurrence was written by Kim et al. [4]. However, unlike in their study, in our study recurrence was reported in only 6 patients. In 4 cases, it was pure DCIS while in 2 invasive breast cancers. In those 6 patients, the high infiltration of FOXP3 + TILs was found to be associated with decreased recurrence-free survival (p = 0.002). However, CD4+ and CD8 + TIL infiltration did not show prognostic significance (p = 0.287). The relationship between the ratios of TILs FOXP3+ /CD8+, FOXP3+/CD4+ and CD4+/CD8+ and tumor recurrence was analyzed and only high FOXP3+/CD8+ and high FOXP3/CD4 + ratios were found to be associated with a decreased recurrence-free survival (p = 0.02 and p = 0.036, respectively) [4]. The findings of Kim et al. [4] concerning the analysis of a small group of primary DCIS and its ipsilateral breast recurrence were in agreement with our observations as regards CD4+, CD8+, and it seems that only such a type of comparison should be performed to study the relationship between immune cells subsets during disease progression.

Syndecan-1 (CD 138) is a transmembrane heparin sulfate proteoglycan, which acts as an extracellular matrix receptor, and is expressed on the surface of mature epithelial cells, precursor B cells and plasma cells [10, 26]. It is involved in many cellular functions, including cell–cell and cell–matrix adhesion. CD-138-clustered antibodies recognize syndecan-1. In some studies of breast cancer, almost all cases of metastatic breast cancer exhibited membranous immune reactivity for CD 138, and all cases of metastatic breast cancer also exhibited stromal reactivity [26]. The majority of epithelial neoplasms, both primary and metastatic, showed reactivity for CD138 for neoplastic cells and frequently also for stromal cells [26]. In other studies, weak expression in malignant ductal cells associated with extensive stromal staining has been described [27]. The functional significance of syndecan-1 expression in the setting of the tumor stroma of breast cancer remains to be elucidated. It has, however, been postulated to have implications for tumor cell growth [26]. As CD 138 interacts with heparin binding growth factors such as fibroblast growth factor-2, its accumulation in tumor stroma might contribute to angiogenesis, cell proliferation and migration, tumor pathogenesis and cell–matrix interactions [26]. Leivonen et al. [28] assessed the prognostic value of the immunohistochemical expression of syndecan-1 in 200 patients with invasive breast cancer. The study revealed that syndecan-1 was expressed in the epithelium in 61% and in the stroma in 67% of tumors. Epithelial expression of syndecan-1 was associated with a negative ER status and stromal syndecan-1 expression with a positive ER status. Stromal expression was found in 71% of ER-positive tumors and 53% ER-negative tumors (p = 0.02). The 10-year breast cancer-specific survival of patients with tumors not expressing stromal syndecan-1 was 83% compared with 66% for those with positive staining. Negative stromal staining was consistently associated with a more favorable survival. Patients with both epithelial and stromal expression had a 10-year survival of 56% as compared to 78% in patients with other expression pattern combinations (p < 0.002) [28]. However, in Cox multivariate analysis, only tumor size and axillary involvement were found to be significant predictors of breast cancer-specific survival. The authors conclude that concomitant expression of epithelial and stromal syndecan-1 seems to identify a group of patients with an unfavorable outcome in invasive breast cancer [28].

Little is known about the importance of immune stromal syndecan-1 in DCIS as a potential prognostic factor of invasive recurrence. In the study by Miligy et al. [10], more CD138 + plasma cells were observed in pure DCIS cases than in DCIS cases with invasion. In our study, the comparison of two subsets of DCIS, with and without recurrence, revealed stromal syndecan-1 CD 138 + plasma cells as the only immune parameter which may help predict recurrence.

To our knowledge, this is the first study in which a potential role of the stromal syndecan-1 as a potential biological marker of poor prognosis of DCIS is attempted to be defined. To confirm or reject our findings, further larger-series studies investigating the composition of immune cell subsets in DCIS are required to understand the role of individual TIL subsets in tumor progression.