Macrophage density is an adverse prognosticator for ipsilateral recurrence in ductal carcinoma in situ

1. Introduction

Evidence of the role the immune microenvironment plays in the development and progression of ductal carcinoma in situ (DCIS) is limited compared to the invasive carcinoma of the breast [[1], [2], [3], [4], [5]]. We previously showed that dense tumor infiltrating lymphocytes (TILs), defined as ≥45% TIL, were associated with an increased risk of ipsilateral recurrence in DCIS [6]. In addition, dense TILs were associated with other parameters of unfavorable behavior including younger age, larger size, high nuclear grade, comedo morphology and estrogen receptor (ER) negativity. Other investigators have similarly demonstrated the association of TILs with high nuclear grade, comedo necrosis, high Van Nuys Prognostic Index (VNPI) and ER and progesterone (PR) negative status [[1], [2], [3], [4], [5]]. It is likely that the explanation for the association of immune response with the histopathologic parameters of aggressiveness and unfavorable outcome lies in the composition of TILs and tumor-associated macrophages (TAM) in the tumor microenvironment [7].

The immune response, or cancer immunoediting, has been classified into three dynamic processes: elimination, equilibrium and escape [8]. The elimination process, which is mounted to survey and eradicate the developing cancer, primarily consists of pro-inflammatory immune response including M1 macrophages, dendritic cells, natural killer cells, CD8⁺ cytotoxic T cell and CD4⁺ T helper 1 cells. In the equilibrium process, the interplay between the immune system and any surviving tumor cell reaches a dynamic equilibrium, in which the tumor may stay dormant for a long time. In the escape process, the surviving tumor cells that have either acquired low antigenicity and insensitivity to immunologic detection and/or have escaped elimination begin to expand in an uncontrolled manner [8]. The escape process shows an anti-inflammatory profile in the tumor microenvironment, characterized by suppressor cells, CD4+/FOXP3+ regulatory T cells, and CD163+ M2 macrophages [1,9].

Exploring the interplay of the DCIS and its microenvironment status may identify immune cellular targets that can potentially be modulated toward achieving a better outcome for patients with DCIS. We sought to investigate the immune status of the DCIS microenvironment and its association with the clinicopathologic parameters in a contemporary cohort of women with pure DCIS.

2. Methods

3. Results

The analytic sample included 67 patients with a DCIS diagnosis in the institutional database (Table 1). The median age was 62 years (IQR = 53 to 66), and the median follow-up was 6.7 years (IQR = 5.3 to 7.8). Among the 67 patients, 13 patients had ipsilateral recurrence within the follow-up period. Of the 13 patients with ipsilateral recurrence, nine (70%) recurred with DCIS, three (24%) recurred with invasive carcinoma and one (8%) recurred with DCIS and microinvasion. The 13 patients with recurrence were on average younger than those without recurrence (mean ± standard deviation: 54.6 ± 13.4 vs. 61.5 ± 11.8, respectively, p = 0.07). Of all the clinicopathologic variables, only the DCIS size and TIL density differed significantly between patients who recurred and those who did not (p = 0.023 and 0.006, respectively).

The distributions of marker variables and cut offs are presented in Table 2. Associations of each dichotomized marker variable with each outcome variable are presented in Table 3. After adjusting for age and TILs density, high CD8 (>70) was significantly associated with negative ER (p = 0.033) and high ki67 (p = 0.014); high CD4 (>90) with negative ER (p = 0.006) and negative PR (p = 0.003); high CD68 (>50) and high CD68/CD163 ratio (>0.46) with ipsilateral recurrence (p = 0.026, p = 0.013, respectively); and high FOXP3 (>6) with negative ER (p = 0.044), negative PR (p = 0.032) and high ki67 (p = 0.01). CD163 and CD8/FOXP3 were not significantly associated with any outcome. These results were confirmed in the sensitivity analyses based on quantile marker variables.

Disease-free Kaplan-Meier survival curves are displayed in Fig. 1. Figs. 1 and ​and22 for dichotomized CD68 and CD68/CD163 ratio variables, respectively. The average time to recurrence for patients with high CD68 (>50) was 64.8 months compared with 78.8 months for those with low CD68 (≤50). The average time to recurrence for patients with high CD68/CD163 ratio (>0.46) was 70.7 months compared with 77.4 months for the low CD68/CD163 ratio group. In Cox proportional hazards ratio models that were adjusted for age and TIL density, high CD68 (>50) and high CD68/CD163 ratio (>0.46) were associated with shorter time to recurrence with hazard ratios (HR) of 4.87 (95% CI: 1.24–19, p = 0.023) and 10.32 (95% CI: 1.34–80, p = 0.025), respectively. We did not see any correlation between her2 positivity and high Ki67 with ipsilateral recurrence (data not shown; adjusted p values: 0.42 and 0.30, respectively).

4. Discussion

The immune infiltrate of the tumor microenvironment can be classified into two interacting components. The first component is the tumor infiltrating lymphocytes (TIL), which, in turn, is a component of the host’s adaptive immunity. TILs can be readily recognized on light microscopy and further subtyped based on their immunohistochemical properties. The second component is the tumor-associated macrophages (TAM), an integral part of the host’s innate immune response. TAMs are the circulating monocytes that are recruited in the tumor microenvironment and differentiated into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages depending on the interplay between TAMs and other inflammatory cells and tumor cells in the tumor microenvironment [8]. M1 macrophages activate type I T helper cells and are tumoricidal. M2 macrophages downregulate immune response and promote angiogenesis. While TAMs are not readily quantifiable on routine histologic sections, they can be highlighted and characterized based on their immunophenotype. CD68 is a pan-macrophage marker recognizing both M1 and M2 macrophages and CD163 specifically highlights M2 macrophages [15,16].

Our analysis demonstrated that after controlling for TIL density and age, high CD68 (>50) and high CD68/CD163 (>0.46) were significantly associated with ipsilateral DCIS recurrence (p = 0.026 and 0.013, respectively; Fig. 3). We also noted a shorter time to ipsilateral DCIS recurrence for both CD68 and CD68/CD163 (Fig. 1, Fig. 2). We did not find a significant association between high CD163 and outcome (p = 0.532). These findings indicate that the high density of TAMs, regardless of their M1/M2 differentiation, is associated with ipsilateral recurrence. Other investigators have demonstrated the association of CD68⁺ macrophage density with adverse prognostic markers including high VNPI, palpability, high nuclear grade, presence of necrosis, and ER and PR negativity in DCIS [2,3]. Our review demonstrates that the only study that reports the correlation between TAM and DCIS outcome is by Chen at al. Which showed that CD68⁺ and CD163+ macrophages were significantly associated with ipsilateral invasive recurrence on multivariate analysis (p = 0.023 and 0.024, respectively) [17]. In this paper the authors demonstrated that while both M1 and M2 macrophages prognosticate ipsilateral invasive recurrence, only CD163+ TAM, M2 macrophage, was associated with all recurrences on multivariate analysis (p = 0.005).

The correlation of high CD68⁺ TAMs and worse outcome in invasive breast cancer has been previously established [15,18,19]. Similarly in DCIS, Campbell et al. showed a significant correlation between CD68⁺ and CD68+/PCNA + TAMs with poor prognostic variable including high VNPI, high nuclear grade, comedonecrosis, and high proliferative index (high Ki67). They did not find the same correlation between their CD68 + MRC1+ cells (M2-type macrophage) and adverse clinicopathologic variables consistent with our results [2]. It appears that the balance between TAM’s pro-inflammatory and anti-inflammatory functional status in the tumor microenvironment is crucial in the biological course of the neoplasm. For example, TAMs promote angiogenesis by producing VEGF and other angiogenic factors and produce growth factors and proteases that enhance tumor progression. TAMs can secrete a variety of proteases including matrix metalloproteinase 7 and 9, which can facilitate the breakdown of basement membrane and tumor cell escape, a process of utmost interest in the progression of DCIS to invasive carcinoma [19]. In other words, dense TAMs can be the harbinger of microinvasion in DCIS. TAMs have also been shown to be capable of releasing ferritin into the breast tumor microenvironment directly stimulating tumorigenesis [20]. The significant correlation of CD68⁺ TAM density and the ratio of CD68+/CD163+ TAM with ipsilateral recurrence in our analysis suggests that the dynamic status of TAM in the DCIS microenvironment leans toward an anti-inflammatory and tumor-promoting milieu in the subset of DCIS with the worse outcome.

We previously demonstrated that high TIL density was a significant predictor of ipsilateral recurrence in patients with pure DCIS [6]. In the current study, we expanded our investigation to TIL subset analysis. Our results showed that after controlling for TIL density and age, the density of CD8, CD4, FOXP3 and the ratio of CD8/FOXP3 were not significantly associated with ipsilateral recurrence. However, high CD8⁺ T cell, high CD4⁺ T cell and high FOXP3+ T regulatory cell density significantly correlated with hormone receptor negativity. In addition, high CD8⁺ T cell and FOXP3+ T regulatory cell density correlated with high ki67 proliferation index. Other investigators have shown that CD8⁺ cytotoxic T cells generally confer a favorable phenotype in ER-negative invasive breast cancer [21,22]. Similarly, CD8⁺ T cell dense microenvironment is typically a protective phenotype in DCIS. Low density of CD8⁺ T cells and an unfavorable CD8+/FOXP3+ ratio have been shown to be associated with increased risk of ipsilateral recurrence [5]. The favorable process of “spontaneous healing” in DCIS also appears to be driven by CD8⁺ T cells [23].

CD4⁺ T cells have been shown to be associated with high grade DCIS, an unfavorable pathologic feature [2]. The association of CD4⁺ T cells with poor pathologic prognosticators including high nuclear grade and hormone receptor negativity in our study may be attributable to the preponderance of T regulatory cells, a subset of CD4⁺ T cells that also co-expresses FOXP3. In our study FOXP3+ cells were significantly associated with high proliferation index, another adverse prognostic phenotype in line with the above observation. In fact, CD4+FOXP3+ regulatory T cell infiltration has been shown to be a poor prognostic indicator in ER + invasive carcinoma [24].

One of the limitations of this study is the use of TMA as opposed to whole tissue section for immunohistochemical studies. However, we mitigated this limiting factor by arraying each case in triplicates. Since our interest was in the microenvironment in the immediate vicinity of DCIS, we believe that our results are minimally affected by examining TMA sections. Another limitation of this study is the relatively small sample size which can be partly attributed to the strict criteria we used to ensure adequate follow-up data and available tissue samples for all patients.

In summary, we conclude that in addition to large size of DCIS and dense TILs, the presence of CD68⁺ TAMs in the DCIS microenvironment is an adverse prognosticator of ipsilateral recurrence. This may have implications as well for identifying those DCIS lesions at risk of progression to invasive cancer.

Declaration of competing interest

The authors have nothing to declare. They have no conflict of interest. There were no ethical issues involved in the preparation of this script.

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