Introduction
FOXP3 is a forkhead box transcription factor, containing a DNA-binding domain that can recruit both transcriptional activator and repressor complexes to target genes [
1]. This transcription factor plays an important role in the development and function of immune regulatory T cells (Tregs), and can be used as a specific biomarker for the identification of Tregs within an inflammatory infiltrate [
2]. Tregs are critical for the maintenance of self-tolerance. There is also mounting evidence that these cells play a central role in immune tolerance to tumor cells by several mechanisms, including inhibiting effector cytotoxic T-cell lymphocytes by reversibly interfering with the release of lytic granules by CD8+ T cells, thereby impeding target cell lysis [
3]. Effective evasion of the immune system by tumor cells is necessary during oncogenesis, tumor progression and metastatic spread. Increased activity of Tregs has been linked with a poor immunological response to tumor antigens and is thought to represent a critical mechanism of immune evasion by tumors. The tumor microenvironment has been reported to contain a rich milieu of molecules capable of increasing the number of FOXP3+ Tregs by several possible mechanisms, including driving CD4+ T-helper cells to develop into FOXP3+ Tregs, recruiting existing FOXP3+ Tregs to the tumor site, and inducing the expansion of resident Tregs. This tumor-induced increase in FOXP3+ Tregs represents a potential barrier to attempts at cancer immunotherapy [
4],[
5].
Studies addressing the prognostic significance of FOXP3+ Tregs have shown conflicting results. The presence of FOXP3+ tumor-infiltrating lymphocytes (TILs) has been reported to be associated with poor clinical outcome in a variety of cancer types, including prostatic, lung, hepatocellular and renal cell carcinomas [
6]-[
10], indicating that cancer patients may benefit from blocking the capacity of tumor cells to recruit Tregs. Conversely, other studies have found that FOXP3+ TILs correlate with favorable prognosis in colorectal, gastric, ovarian and head and neck carcinomas [
11]-[
15]. These discrepant prognostic associations of FOXP3+ TILs reflect the complexity of biological processes affecting the host immunological response to tumoral tissue – in some tumors, immune infiltrates are recruited by tumor cells and facilitate tumor spread, whereas in other tumors immune infiltrates reflect a host anti-tumor reaction. The type of T cell present may help distinguish between these types of responses, but this requires subtyping of TILs into regulatory (FOXP3+) and cytotoxic (CD8+) populations. For example, we recently showed that the presence of CD8+ cytotoxic T-cell infiltrates in breast cancer is a good prognostic factor in basal breast cancers, but not in the other intrinsic molecular subtypes of breast cancer [
16].
Even within breast cancer, the prognostic significance of FOXP3+ TILs has been widely debated. Recent studies have reported that FOXP3+ T-cell infiltration is associated with poor clinical outcome [
17]-[
22], whereas others found no significant prognostic role for FOXP3+ infiltration in a large series of breast cancers [
23]. Indeed, some recent evidence suggests that FOXP3+ TILs could actually be a favorable survival indicator in certain subgroups. A recent study, using a cohort of 175 estrogen receptor (ER)-negative breast cancers found that FOXP3+ TILs were positively correlated with the concurrent presence of CD8+ TILs, and were an independent good prognostic factor in ER-negative breast cancer [
24]. Another recent study reported that FOXP3+ TILs were a significant and independent factor associated with improved overall survival and progression-free survival in triple-negative breast cancer [
25]. The properties of FOXP3+ TILs may be affected by the tumor microenvironment, thus the prognostic value of FOXP3+ TILs could possibly be influenced by molecular subtype and interactions with other immune cells. To date, few studies have had sufficient power to investigate the prognostic effect of the interaction of FOXP3+ TILs with molecular subtypes or different types of immune response in breast cancer.
We implemented this study to evaluate the prognostic significance of FOXP3+ TILs, in a population-based breast cancer cohort with long-term follow-up and detailed biomarker data defining the main intrinsic molecular subtypes and cytotoxic T-cell infiltrates in breast tumor tissues.
Discussion
The infiltration of tumors by regulatory T cells (defined as FOXP3+ TILs) has been reported to be associated with patient survival in a variety of cancers, but its prognostic value remains controversial. We evaluated the prognostic effect of FOXP3+ TILs in breast cancer, using a large population-based breast cancer cohort with long clinical follow-up. In addition to assessing the independent prognostic value of FOXP3+ TILs, adjusted by conventional clinicopathological confounding factors, we also investigated the prognostic effect of the interaction between regulatory and cytotoxic T-cell infiltrates (defined as CD8+ TILs) in different molecular subtypes of breast cancer, a novel analysis made possible by the large sample size and availability of concurrent biomarker data. Results from this study demonstrate that, in breast cancer, the presence of high levels of FOXP3+ TILs is associated with young age, high grade, positive nodal status, concurrent CD8+ T-cell infiltrates, and ER negativity (including both the HER2+/ER– and core-basal subtypes). In ER + breast cancer, high FOXP3+ TILs were significantly associated with poor clinical outcome. When ER + tumors did not display concurrent CD8+ lymphocyte infiltration, patients with high levels of FOXP3+ TILs had 30% higher cumulative death rates than those with low FOXP3+ TILs. However, in ER– group, the prognostic effect of FOXP3+ TILs differed by intrinsic subgroup, depending on the presence of CD8+ T-cell infiltration. In HER2+/ER– breast cancer, the multivariate analysis indicates that high FOXP3+ lymphocyte infiltration is a significant, independent and favorable prognostic factor in patients having tumors with CD8+ T-cell infiltrates. In this subgroup, those with high levels of FOXP3+ TILs had 52% higher probability of survival than those with low FOXP3+ TILs. However, in the core-basal subgroup, although high levels of FOXP3+ lymphocyte infiltration was associated with improved survival in general, the presence of CD8+ T-cell infiltrates appeared to be the stronger and more important favorable prognostic factor.
The prognostic associations of FOXP3+ TILs could be affected by tumor microenvironment, including tumor site, histologic and molecular subtype, and different types of immune response, all of which may interactively influence clinical outcome of cancer patients. In general, patients with ER + breast cancers experience better clinical outcome than those with ER– tumors, but not all ER + breast cancer patients have good survival. It was reported that FOXP3+ TILs were associated with poor clinical outcome in ER + breast cancer [
17],[
23]. The multivariate analysis from our study confirms that, in ER + breast cancer, FOXP3+ regulatory TILs are an indicator for poor survival, at least in those tumors lacking concurrent cytotoxic CD8+ T-cell infiltration. One possible explanation consistent with these findings is that FOXP3+ TILs reflect tumor-induced immune evasion in ER + breast cancers. Although patients with ER– tumors usually have poor survival, some biological signatures serve as favorable prognostic indicators among ER– breast cancers. Studies with clinical and gene expression data demonstrate that an immune response is significantly associated with improved prognosis among ER– breast cancers [
38]-[
41]. In our study, FOXP3+ TILs were found to be a favorable indicator of survival in ER– breast cancer, which is also consistent with the findings from other recently published studies [
24],[
25]. Cause and effect cannot be determined from this type of association study, but for the HER2+/ER– group at least one interpretation consistent with the data is that FOXP3+ TILs are induced secondarily by a robust antitumor CD8+ TIL response.
Until now, comparatively few studies have been powered to assess if different categories of tumor-infiltrating lymphocytes influence clinical outcomes in different breast cancer molecular subtypes. Our study indicates that the infiltration of FOXP3+ lymphocytes not only has distinctive prognostic associations within different subtypes of breast cancer, but also interacts with cytotoxic T-cell infiltrates in their association with patient survival. Our multivariate analyses stratified for presence or absence of CD8+ T-cell infiltration demonstrated that the favorable prognostic effect of FOXP3+ TILs in ER– breast cancer was only significant and independent in those having HER2+/ER– tumors with concurrent CD8+ T-cell infiltrates. In the core basal subtype, the favorable prognostic effect associated with a TIL immune response may be primarily due to CD8+ T-cell infiltration, which may be counteracted when FOXP3+ Tregs are also present. Despite our large sample size and event rate with long follow-up, these subgroup analyses still have limited power, and as a retrospective cohort analysis will require external validation or incorporation into meta-analyses to increase the level of evidence for these results.
The prognostic significance of immune responses in breast cancer is controversial. Most of the studies reporting that FOXP3+ TILs are an indicator of poor prognosis in breast cancer applied unstratified survival analyses [
17],[
19] and might be expected to largely reflect the majority ER + population, possibly confounded by an opposite association in ER– cases. Recent studies, using large breast cancer cohorts, have consistently demonstrated that cytotoxic T-cell infiltration plays different prognostic roles in different molecular subtypes [
16],[
35],[
42]. To our knowledge, ours is the first study with sufficient power to evaluate the prognosis of these interactions stratified by subtype and cytotoxic vs. regulatory lymphocyte infiltration. Some studies have applied ratios of CD8+: FOXP3+ TILs to evaluate the prognostic effect of immune response in breast cancer, and reported that those with a ratio of CD8+: FOXP3+ TILs ≥1 had better survival than those with ratios <1 [
21],[
24],[
43]. The results from our study suggest that both FOXP3+ and CD8+ T-cell infiltration have distinctive prognostic implications in different molecular subtypes of breast cancer. A simple ratio between FOXP3+ and CD8+ TILs may not be an appropriate universal prognostic indicator, across biologically different breast cancer subtypes.
Inconsistency in defining and measuring tumor infiltrating lymphocytes may also underlie apparently discrepant research results. In this study, we used specific immunohistochemistry with a widely used mouse monoclonal anti-human FOXP3 antibody to detect regulatory intratumoral and stromal TILs in breast tumor tissue. Both ROC curve and X-tile methods were applied to define the optimal cutoff points of FOXP3+ TILs for the survival analyses. These findings apply to the TMA platform but would need modification and re-validation to be applied to whole sections. To confirm the prognosis of FOXP3+ TILs in breast cancer, we also evaluated the association of FOXP3+ iTIL with relapse-free survival; results were very similar to those using breast cancer-specific survival as the outcome (Tables S3-S5 in Additional file
1). Furthermore, we tested the correlation of FOXP3+ sTILs and tTILs with patient clinical and pathological characteristics. As with the primary assessment of intratumoral lymphocytes, results demonstrated positive correlations between presence of high numbers of FOXP3+ sTIL and tTIL with young age, high grade, positive nodal status, CD8+ T-cell tumor infiltration, ER negativity, and with HER2+/ER– and core basal subgroups (Table S6 in Additional file
1).
Although we believe that the measurement of FOXP3+ TILs and the evaluation of the association of FOXP3+ TILs with breast cancer patient survival are reliable and form a consistent picture with other reports, there are some limitations in this study. Studies suggest that FOXP3 represents the most specific marker for Treg cells, but some rare subtypes of cytotoxic lymphocytes may also express FOXP3 [
17],[
44]. This study only assessed the interaction between FOXP3+ and CD8+ T-cell infiltration in breast cancer; further studies need to be done to differentiate other types of chronic inflammatory infiltration. Studies have reported that pre-existing immune responses may fortify the effect of chemotherapy [
45],[
46]. TILs could be a favorable predictive indicator for targeted trastuzumab therapy [
47],[
48]. The patients in our study predate the trastuzumab era, and the types of endocrine and chemotherapy given would be considered outdated by contemporary standards of care. An exploratory multivariate analysis (Table S7 in Additional file
1) found that pre-existing FOXP3+ Treg infiltration might be a favorable factor for the group of patients treated with conventional chemotherapy (for BCSS: HR = 0.67, 95% CI = 0.50 to 0.89,
P = 0.006; for RFS: HR = 0.69, 95% CI = 0.53-0.90,
P = 0.005), but treatment was not randomly assigned in our study cohort. Formal prospective-retrospective studies of randomized trials [
49], using locked-down assay and interpretation methodology based on cohort studies such as ours, would be the next step to determine, with a higher level of evidence, the relationship between chemotherapy, immune infiltrates and clinical outcome of cancer patients.
Competing interests
The authors declare that they have no competing interests.