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Prognostic value of tumor-infiltrating Foxp3+ regulatory T cells in patients with breast cancer: a meta-analysis

Purpose: The prognostic value of tumor-infiltrating Foxp3+ regulatory T cells (Tregs) in breast cancer remains controversial. Therefore, we performed this meta-analysis to determine the impact of Foxp3+ Tregs infiltration on survival outcomes. Methods: Relevant literature was retrieved from Pubmed, Web of science and Cocohrane until May 30, 2016. Meta-analysis was performed using hazard ratios (HRs), odds ratio (OR) and 95 % confidence intervals (CI) as effect measures. Results: Fourteen studies (10,259 patients) were included. Meta-analysis showed that high Foxp3+ Tregs infiltration was correlated with high histological grade (OR= 2.96, 95%CI [2.03-4.31]), estrogen receptor (ER) negativity (OR= 0.38, 95%CI [0.23-0.60]), human epidermal growth factor receptor type 2 (HER2) positivity (OR=2.43, 95%CI [1.69-3.51]). The detection of FOXP3+ Tregs was significantly associated with recurrence-free survival (RFS) of patients (HR = 1.58, 95 % CI [1.03-2.44]). Conclusion: Our meta-analysis suggests that high Foxp3+ Tregs infiltration is associated with poor RFS in breast cancer patients and predicts histological grade, estrogen receptor and HER-2 status.


Introduction
Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer-related mortality among women worldwide [1,2]. Despite recent progress in breast cancer diagnosis and therapy, the prognosis of breast cancer patients is still poor. Local tumor microenvironment, including tumor cells, extracellular substrates, cytokines and tumor-infiltrating immune cells play an essential role in tumor formation, growth, invasion, and metastasis [3][4][5]. Regulatory T cells (Tregs) are a subgroup of CD4 + T helper cells that suppress T cell immunity. Forkhead box protein P3 (FoxP3) is a transcription factor imperative and sufficient for the induction of immunosuppressive function of Tregs and could be the most specific marker for Tregs in tumors [6].
Due to the ability to inhibit anti-tumor immunity, high level of tumor-infiltrating Foxp3+ Tregs is expected to be associated with poor prognosis. However, recent studies have challenged this idea and shown that Foxp3+Tregs could improve survival in some tumors [7][8][9]. To resolve the contradiction, in this study we performed a meta-analysis to investigate the correlation of Foxp3+Tregs and the survival of breast cancer patients.

Search strategy
Pubmed, Web of science, Cocohrane (last search updated in May 30, 2016)

Exclusion criteria
(1) Reviews, meta-analysis and conference abstracts were excluded. (2) Animal or cell line studies. (3) Peripheral blood samples or Foxp3 expressed on tumor cells.

Data extraction
Two investigators independently extracted the data from each study, including the first author's surname, geographical location, sample size, publication date, the source of the subjects, protein detection method, median follow-up time and survival data, the discrepancy was resolved by discussion.

Statistical analysis
The heterogeneity was assessed by X 2 test and the I 2 statistic of inconsistency [11,12]. Statistically significant heterogeneity was defined as X 2 P < 0.1 or I 2 > 50%. We conducted subgroup analysis by stratifying Tregs location and geographical location. For the pooled analysis of the correlation between FoxP3+ Tregs infiltration and clinicopathological features (Tumor size, histological grade, Lymphnodes metastasis, ER and HER-2 status), odds ratios (ORs) and their 95% CIs were combined to estimate the effect. We performed sensitivity analysis to evaluate the influence of single study on the overall outcome. In addition, we performed funnel plots and Begg's linear regression test to examine publication bias [13]. All analysis was performed using STATA version 12.0.
We extracted RFS data from 8 of the 14 articles and OS data from 11 of the 14 articles. These studies evaluated patients from East Asia, Europe, Middle East and North America, including 3 from China, 2 from Japan, 2 from Korea, 2 from Netherlands, 2 from UK, 1 from Turkey and 2 from Canada. All the included studies evaluated FOXP3+ Tregs with immunohistochemical method. The 14 studies included 10,259 patients, the sample sizes ranged from 86 to 3,277. Six of these studies enrolled less than 200 patients and eight studies included more than 200 patients. The NOS scores of these studies ranged from 5 to 8, thus the quality of all included studies was good. Table 1

Impact of FOXP3+ Tregs on survival outcomes
As shown in Figure 2, the pooled results of eight studies that assessed RFS showed that patients with high overall Foxp3+ T cells infiltration had significantly decreased RFS compared to patients with low/negative Foxp3+ Tregs infiltration in the random effects model (combined HR 1.58, 95% CI 1.03-2.44) and presented a significant degree of heterogeneity (I²=77.5%, p<0.0001). Subgroup analysis based on geographical location showed similar results to overall analysis in Asian group patients

Sensitivity analysis
By sensitivity analysis we found similar HRs and 95% CIs if we omitted any single study, indicating that the results of RFS and OS were relatively stable (Figure 4 and 5).

Publication bias
For RFS, the evaluation of publication bias by Bgger tests (p=0.386, Figure 6) showed no publication bias for all included studies. Egger's linear regression did not reveal obvious publication bias (P=0.057). For OS, Bgger's funnel plot did not show obvious asymmetry (P = 0.119, Figure 7), and Egger's linear regression did not reveal obvious publication bias (P = 0.069).

Discussion
Tumor immunity has big impact on clinical outcome of cancer patients [28]. Tregs play an important role of immunosuppression by regulating the balance between tolerance and rejection of self and altered self and secreting cytokines such as TGF-b, IL-4 and IL-10 [29]. Tregs markers such as GITR, OX-4, CTLA-4, CD127 and transcription factor FoxP3 have been reported, and FoxP3 is regarded as the most specific marker [30,31]. Emerging evidence suggests that Tregs play an essential role in immune evasion mechanisms of cancer [32][33][34][35]. However, prognostic significance of FOXP3+ Tregs in breast cancer patients is still unclear. A study showed that Foxp3+Tregs were associated with good outcome of breast cancer patients [12]. Nevertheless, other studies suggested that Foxp3+Tregs were correlated with poor prognosis of breast cancer [17,20]. Insignificant association between Foxp3+Tregs and prognosis of breast cancer patients was also reported [15,16]. The inconsistency may be caused by different genetic backgrounds, chemotherapy agents and surgical procedures. A recent review suggested that Tregs are equipped with cytotoxic potential which may directly kill effector T cell [36]. This may explain the relationship between infiltration of Foxp3+ Tregs and poor RFS of breast cancer patients. In addition, our data showed that high infiltrated Foxp3+ Tregs was associated with a worse OS. However, the results were not statistically significant. As we know, many kinds of factors may affect overall survival rate of patients. Thus Foxp3+ Treg alone may be not enough to predict OS of breast cancer patients.
In this meta-analysis, we investigated the association between Foxp3+Tregs and clinical outcome of breast cancer patients. Combining the data of 10,259 patients from 14 studies, our analysis revealed that high overall Foxp3+Tregs infiltration was positively correlated with high histological grades, ER negativity and Her-2 overexpression, and short RFS. However, FOXP3+Tregs infiltration was not associated with OS of breast cancer patients. Subsequently, sensitivity analysis confirmed that our results were stable. Funnel plot analysis revealed no publication bias. The relationship between Foxp3+Tregs and poor RFS may be explained by the immunosuppressive function of Foxp3+Tregs in breast cancer. On the other hand, the relationship between Foxp3+Tregs and OS may be explained by that Foxp3+Tregs infiltration alone is not enough to predict the OS of breast cancer patients. The ratio between effector T cells and Tregs may serve as a better independent prognostic factor for OS of breast cancer patients [37].
We acknowledge that our meta-analysis has several limitations. First, we only selected the studies published in English. Second, the test for heterogeneity of included studies was significant (I 2 = 77.5%, p<0. 0001 and I 2 =73%, p<0.001). We employed subgroup analysis and sensitivity analysis to find out the source, but the results were negative. Despite these limitations, the selection process of the eligible articles was strict and all included studies had high quality. Moreover, 10,259 patients were included in our meta-analysis, which made our results convincible.
In conclusion, our meta-analysis indicates that the presence of high levels of FOXP3+ Tregs is associated with poor RFS for breast cancer patients and predicts histological grade, estrogen receptor and HER-2 status.