The adaptive immune system is known to play a major role in the control of tumor progression in different types of cancer. Indeed, tumor infiltration by CD8+
T-cells has been shown to represent an important prognostic factor in melanoma [7
], and, more recently, in colorectal cancers [2
Early studies have suggested a favorable prognostic effect of lymphocyte infiltration in breast cancers [34
]. More recently, infiltration by CD3+
T-cells has been suggested to predict responsiveness to neoadjuvant treatment in these tumors [25
]. Furthermore, a predictive effect of breast cancer infiltration by FOXP3+
cells has also been reported [35
]. Breast cancers, however, comprise histologically different tumor entities characterized by molecular specificities and differential prognosis [36
]. The expression of hormone receptors and her2/neu also represent important factors in the biology of breast cancers and in its prognosis [36
]. Most importantly, phenotypes and location of tumor infiltrating lymphocytes are emerging as important issues in cancer immunobiology [7
]. Therefore, a thorough assessment of the immunobiological relevance of lymphocyte infiltration in breast cancer needs to accurately take into account these parameters.
Our data, deriving from the study of a large cohort of cases, including > 1000 specimens, are consistent with a significantly different pattern of lymphocyte infiltration in ductal and lobular breast cancers. In the lobular histological type, there is a lower lymphocytic infiltration than in ductal cancers, particularly regarding CD4+ and FOXP3+ cells. Furthermore, in lobular breast cancers, lymphocyte infiltration is not correlated with tumor grade and expression of hormonal receptors and it has no prognostic relevance.
In contrast, in ductal cancers, increased infiltration by CD4+
lymphocytes correlates significantly with histological grade, and ER loss. Her2/neu over-expression in ductal cancers is also significantly associated with increased numbers of FOXP3+
infiltration. Puzzlingly, loss of PR expression appears to be associated with a decrease of CD4+
infiltrate. In agreement with previous reports [27
], we also found that FOXP3+
infiltration is significantly increased in triple negative ductal, but not in lobular breast cancers.
Whereas the molecular background underlying these effects is unclear, they do impact the clinical course of the disease, since in ductal cancers, high infiltration by CD4+ T-cells is associated with a significantly more severe prognosis, albeit only in univariate analysis.
On the other hand, in both ductal and lobular cancers only a modest infiltration by IL-17 producing cells was detectable.
Ductal breast cancers are more compactly growing tumors, sometimes characterized by a broad and pushing border, whereas lobular carcinomas mainly show indian file pattern, smoothly infiltrating the surrounding tissue. Interestingly, the detection of increasing numbers of TILs in high grade tumors with pushing borders was described earlier in medullary breast cancers [23
]. In our study, higher counts of TILs are significantly (p
< 0.001) associated with more aggressive tumor features such as loss of estrogen receptor, higher tumor grade (G3), or her2/neu over-expression in ductal breast cancers. More aggressive tumors are growing faster and may therefore present more necrotic areas while producing stroma damage possibly related to local hypoxia [39
]. Indeed, increased numbers of CD4+
cells under hypoxic condition could be shown in several studies [43
]. Therefore, the growth dynamics of the tumor could play a role in inducing lymphocyte infiltration.
FOXP3 represents a typical, although not exclusive, marker of regulatory CD4+
T-cells. Tumor infiltration by T-cells expressing this marker has been associated to severe prognosis in different tumors, including ovarian and lung cancers [8
]. However, in colorectal cancers FOXP3+
T-cell infiltration has been found to correlate with significantly improved prognosis by us and others [44
]. Our data clearly document that, although numbers of FOXP3+
lymphocyte infiltration in ductal breast cancer are significantly associated with unfavourable clinico-pathological features, this marker alone does not appear to represent a prognostic marker. However, a high ratio (> 1) of total FOXP3+
TILs was independently associated with a better overall survival, thereby suggesting that FOXP3+
cells other than CD4+
T lymphocytes could be involved in the elicitation of the favorable prognostic effects. It is of note, that although FOXP3 still represents one of the most reliable Treg markers, it is known to be expressed by activated T-cells as well [8
]. Indeed, FOXP3 has been shown to be expressed, albeit transiently, in activated CD8+
T cells [46
], in tonsillar suppressive CD8+
T cells [47
], and even in tumor cells [48
Contrasting data have been reported regarding FOXP3 expression in tumor cells. In a mouse model FOXP3 could be identified as a X-linked tumor suppressor gene in breast cancer [49
], but others have failed to detect expression of this gene in non hematopoietic tissues [50
]. FOXP3 expression has also been reported in human breast cancer cells [26
]. Other groups however, did not confirm these findings and only detected FOXP3 expression in breast cancer infiltrating lymphocytes [35
]. Furthermore evidence has been reported that localization and activation status of FOXP3 positive cells might play a prognostic role in breast cancer [53
]. Still unclear is the background underlying these discrepancies, possibly related to the use of different reagents and staining protocols. In our studies however, in keeping with previous results [55
] expression of FOXP3 in breast cancer cells was found to be negligible while it was usually detectable as nuclear staining of TIL.
Accordingly, CD3 and CD163 specific staining data indicate that tumor infiltration by FOXP3+ cells is highly correlated with infiltration by CD3+ cells. Further studies are warranted to clarify nature, origin and functions of FOXP3+ cells associated with improved survival in ductal breast cancer. Nevertheless, our data suggest that FOXP3 expression might reflect an activated state of specific T cell subsets.
Our study indicates that ductal and lobular breast cancers are characterized by a significantly different pattern of lymphocyte infiltration. Notably, in ductal cancers, total and, in particular, intratumoral lymphocyte infiltration is significantly associated with higher histological grade and severe prognosis, although not independently from known prognostic factors.
Further research is warranted to clarify whether these features are related to differential growth patterns of these tumor types, or to a differential immunogenicity of these tumors. Alternatively, variable tumor microenvironments might differentially favour lymphocyte chemoattraction and expansion.