The forkhead box protein 3 (FOXP3) transcription factor is highly expressed in tumor cells as well as in regulatory T cells (Tregs). It plays a tumor-enhancing role in Tregs and suppresses carcinogenesis as a potent repressor of several oncogenes. The clinical prognostic value of FOXP3 expression has not yet been elucidated. In this study, immunohistochemistry was used to investigate the prognostic significance of FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in breast cancer patients. Of the 100 tumor specimens obtained from primary invasive breast carcinoma, 63 and 57% were evaluated as FOXP3+ tumor cells and as being highly infiltrated by FOXP3+ lymphocytes, respectively. Although FOXP3 expression in tumor cells was of no prognostic significance, FOXP3+ lymphocytes were significantly associated with poor overall survival (OS) (n=98, log-rank test P=0.008). FOXP3 exhibited a heterogeneous subcellular localization in tumor cells (cytoplasm, 31%; nucleus, 26%; both, 6%) and, although cytoplasmic FOXP3 was associated with poor OS (P= 0.058), nuclear FOXP3 demonstrated a significant association with improved OS (P=0.016). Furthermore, when patients were grouped according to their expression of tumor cytoplasmic FOXP3 and lymphocyte FOXP3, there were notable differences in the Kaplan-Meier curves for OS (P<0.001), with a high infiltration of FOXP3+ lymphocytes accompanied by a cytoplasmic FOXP3+ tumor being the most detrimental phenotype. These findings indicated that FOXP3 expression in lymphocytes as well as in tumor cells may be a prognostic marker for breast cancer. FOXP3 in tumor cells may have distinct biological activities and prognostic values according to its localization, which may help establish appropriate cancer treatments.
Forkhead box protein 3 (FOXP3) is a member of the forkhead/winged-helix family of transcription factors involved in the regulation of the development and function of the immune system (
FOXP3 plays a crucial role in the generation of immunosuppressive CD4+CD25+ regulatory T cells (Tregs), which induce immune tolerance to antigens (
FOXP3 protein expression was initially considered to be restricted to the lymphocyte lineage. However, its expression has been demonstrated in various types of non-hematopoietic cells, including human tumor cells (
In a different context of tumor-expressed FOXP3, Hinz
FOXP3 is constitutively expressed in the nucleus of human Tregs (
A total of 100 adult females with primary invasive breast carcinoma who underwent breast surgery at our institution (Kurume University Hospital, Kurume, Japan) between 1995 and 2005 and who had not received neoadjuvant chemotherapy, were enrolled in the present study. Hematoxylin and eosin (H&E)-stained histological sections from each patient were analyzed for biological parameters and histological grading was performed using the Nottingham-combined histological grade [Scarff-Bloom-Richardson (SBR) grading system] (
The estrogen receptor (ER) and progesterone receptor (PgR) status were analyzed immunohistochemically on formalin-fixed, paraffin-embedded tumor sections, using ER (clone SP1) and PgR (clone 1E2) antibodies at a dilution of 1:100 and the iVIEW system (Ventana Medical Systems, Tucson, AZ, USA). Labeling was detected using the Ventana BenchMark XT automat (Ventana Medical Systems). The arrays were counterstained with hematoxylin. The HercepTest scoring method with the 4B5 antibody (Ventana Medical Systems) was used to determine the HER2 status, with a score of 3+ or 2+ with fluorescent
FOXP3 expression was immunohistochemically analyzed using rat anti-human FOXP3 monoclonal antibody clone ab22510 (Abcam, Cambridge, UK). Paraffin-embedded tissue samples were cut into 4-μm sections and examined on a coated glass slide. Intrinsic peroxidase activity was blocked by treatment with peroxidase-blocking reagent (DakoCytomation, Glostrup, Denmark) for 5 min. The specimens were boiled in a microwave for 30 min in 1 mmol/l EDTA (pH 9.0) target retrieval solution (DakoCytomation), to recover the antigens. After washing in Tris-buffered saline (TBS; DakoCytomation) for 10 min, the FOXP3 antibody was diluted 1:600 and applied to the specimens. Histological specimens were incubated at 4°C overnight, washed in TBS for 15 min and incubated with labeled polymer-horseradish peroxidase (HRP) secondary antibody (ChemMate Envision kit; DakoCytomation) for 30 min at room temperature. After washing in TBS for 10 min, the slides were visualized using 3,3′-diaminobenzidine.
FOXP3 expression was evaluated independently by two authors (M.T and M.K), who were blinded to the clinico-pathological data. Discrepancies were reviewed jointly and a consensus was reached. The staining intensity of FOXP3-positivity (FOXP3+) within the tumor-cell cytoplasm was scored as weak (1+) or strong (2+) (
Overall survival (OS) was defined as the time period between the time of surgery and the time of death from any cause. Patients who were alive at the last contact attempt were regarded as censored cases at this time point. Relapse-free survival (RFS) was defined as the time period from the time of surgery until progressive disease was confirmed by magnetic resonance imaging (MRI) or computed tomography (CT), or until death from any cause. Patients without progressive disease were regarded as censored cases at the date of their last CT or MRI examination.
For tumor-cell cytoplasm FOXP3 expression, a score of 0 was defined as negative and scores of 1+ or 2+ as positive. For lymphocyte FOXP3 expression, scores of 0 and 1+ were defined as negative (absent or low infiltration) and scores of 2+ and 3+ as positive (high infiltration). These definitions accounted for the median score and minimized the difference between the number of patients classified as negative and those classified as positive. For tumor nuclear FOXP3 expression, ≥30% was defined as positive and <30% as negative from a statistical viewpoint. In the Cox regression model with a binary explanatory variable representing positive or negative with various cut-off points, we selected the value maximizing the profile partial likelihood, i.e., we selected the cut-off value that provided the best fit to the OS data using various classifications. Associations between FOXP3 expression in tumor cells and lymphocytes and between FOXP3 expression and clinicopathological factors were examined with the Fisher’s exact test. Survival functions for OS and RFS were estimated with the Kaplan-Meier method and compared with the log-rank test. Cox regression analysis was performed to examine whether FOXP3 expression was associated with OS or RFS following adjustment for possible confounding factors. Clinicopathological characteristics significantly associated with FOXP3 expression were included in the Cox regression for adjustment.
Statistical analyses were conducted with SAS version 9.2 (SAS Institute Inc., Cary, NC, USA) and R version 2.9.0. P<0.05 was considered to indicate a statistically significant difference.
Of the 100 tumor specimens immunostained for FOXP3, 63 (63%) and 57 (57%) were evaluated as positive for expression in tumor cells and TILs, respectively. FOXP3 was expressed in the nucleus of lymphocytes, representing Treg infiltration, whereas a heterogeneous subcellular localization of FOXP3 was observed in tumor cells (i.e., the cytoplasm and/or nucleus;
Prognostic analysis was performed using the 98 patients whose clinical outcome was monitored. Univariate analysis of clinicopathological characteristics indicated that high tumor grade (III) and ER negativity were significantly associated (P<0.05) with mortality (OS), whereas no significant prognostic value for OS was observed when the other factors were assessed (
Kaplan-Meier curves confirmed that FOXP3 expression localized in the cytoplasm or nucleus of tumor cells was associated with worse (log-rank test, P=0.058) or improved (log-rank test, P=0.016) OS, respectively (
Multivariate analysis of the covariates with P<0.05 in
FOXP3+ Tregs are immunosuppressive, therefore, their abundance in tumor infiltrates is associated with an unfavorable clinical outcome. Several previous studies reported that increased infiltration of FOXP3+ lymphocytes in the tumor microenvironment was associated with poor prognosis in cancer patients (
The present study has demonstrated that FOXP3 localization in breast cancer was crucial to predicting clinical outcome. Zuo
The underlying mechanism(s) by which the expression of tumor FOXP3 affects prognosis require further investigation. Zuo
Accumulating evidence indicates that FOXP3 coordinates with multiple transcriptional regulators and its localization may depend on its molecular partners (
A previous study suggested that tumor-expressed FOXP3 triggers a mechanism for the immune evasion of tumor cells (
Our data suggested that FOXP3 expression in tumor cells and TILs may be an effective prognostic marker in breast cancer patients and that FOXP3 localization in tumor cells is an important determinant of prognosis. FOXP3 may provide distinct biological activities and prognostic values according to its localization. However, multivariate analysis demonstrated that FOXP3 expression in TILs, unlike that in tumor cells, was an independent prognostic factor for OS (
This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the ‘High-Tech Research Center’ Project for Private Universities. A matching fund subsidy was provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Immunohistochemical forkhead box protein 3 (FOXP3) staining in breast cancer. Representative images of FOXP3 expression in (A) the cytoplasm (magnification, ×100) and (B) the nucleus (magnification, ×400) of tumor cells and (C) the lymphocytic infiltrate (magnification, ×400). (D) Hematoxylin and eosin (H&E) staining intensity of tumor-infiltrating lymphocytes (TILs) (magnification, ×200). The number of specimens in each graded group is indicated in parenthesis. A score of 1+/2+ in (A) or 2+/3+ in (C) was defined as positive for tumor-cytoplasmic FOXP3 or lymphocyte FOXP3 (i.e., high infiltration of FOXP3+ lymphocytes).
Kaplan-Meier curves for overall survival (OS) associated with forkhead box protein 3 (FOXP3) expression in breast cancer. Kaplan-Meier curves in two groups divided into (A) FOXP3-positive (+/−, −/+ and +/+, cytoplasmic/nuclear expression) and -negative (−/−), (B) cytoplasmic FOXP3-positive (+/− and +/+) and -negative (−/+ and −/−) and (C) nuclear FOXP3-positive (−/+ and +/+) and -negative (+/− and −/−) expression in tumor cells. Intensities of (D) tumor-infiltrating lymphocytes (TILs) and (E) FOXP3+ lymphocytes were stratified as follows: absent-low, intermediate and high infiltration in (D); negative (absent-low) and positive (high infiltration of FOXP3+ lymphocytes) in (E). Kaplan-Meier curves in the four groups according to infiltration of (F) FOXP3+ lymphocytes and cytoplasmic or (G) nuclear FOXP3 in tumor cells. Number of specimens in each group is shown in parenthesis. P-values were calculated using the log-rank test. neg, negative; pos, positive.
Clinicopathological characteristics of breast cancer patients.
Characteristic | n (%) |
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Total no. of patients | 100 |
Age (years) | |
≤50 | 29 (29) |
>50 | 71 (71) |
Tumor size (cm) | |
≤2.0 | 59 (59) |
>2.0 | 41 (41) |
Axillary nodal status | |
Positive | 45 (45) |
Negative | 47 (47) |
Resection not performed | 8 (8) |
Tumor grade | |
I and II | 77 (77) |
III | 23 (23) |
HER2 | |
Positive | 23 (23) |
Negative | 77 (77) |
ER | |
Positive | 56 (56) |
Negative | 44 (44) |
Triple-negative |
21 (21) |
ER−/PgR−/HER2− phenotype. HER2, human epidermal growth factor receptor 2; ER, estrogen receptor.
Localization of FOXP3 expression.
Tumor-cell cytoplasm | Tumor-cell nucleus | |||||
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FOXP3+ | FOXP3− | FOXP3+ | FOXP3− | |||
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Patient no. (%) | Patient no. (%) | P-value | Patient no. (%) | Patient no. (%) | P-value | |
Total patient no. | 37 | 63 | 32 | 68 | ||
Tumor-cell cytoplasm | ||||||
FOXP3+ | 6 (18.8) | 31 (45.6) | 0.014 | |||
FOXP3− | 26 (81.3) | 37 (54.4) | ||||
Tumor-cell nucleus | ||||||
FOXP3+ | 6 (16.2) | 26 (41.3) | 0.014 | |||
FOXP3− | 31 (83.8) | 37 (58.7) | ||||
Lymphocytes |
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FOXP3+ | 23 (62.2) | 34 (54.0) | 0.531 | 11 (34.4) | 46 (67.6) | 0.002 |
FOXP3− | 14 (37.8) | 29 (46.0) | 21 (65.6) | 22 (32.4) |
Evaluated by Fisher’s exact test.
FOXP+, high infiltrate; FOXP3−, absent-low infiltrate of FOXP3-expressing lymphocytes. FOXP3, forkhead box protein 3.
Frequency of patient clinicopathological characteristics according to FOXP3 expression.
Tumor-cell cytoplasm | Tumor-cell nucleus | Lymphocytes |
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FOXP3+ | FOXP3− | FOXP3+ | FOXP3− | FOXP3+ | FOXP3− | ||||
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Characteristic | n/total (%) | n/total (%) | P-value | n/total (%) | n/total (%) | P-value | n/total (%) | n/total (%) | P-value |
Age, years (>50 years) | 24/37 (64.9) | 45/63 (71.4) | 0.510 | 22/32 (68.8) | 47/68 (69.1) | 1.000 | 38/57 (66.7) | 31/43 (72.1) | 0.664 |
Tumor size (>2 cm) | 21/37 (56.8) | 21/63 (33.3) | 0.035 | 11/32 (34.4) | 31/68 (45.6) | 0.386 | 25/57 (43.9) | 17/43 (39.5) | 0.688 |
LN metastasis | 21/31 (67.7) | 24/61 (39.3) | 0.015 | 13/32 (40.6) | 32/60 (53.3) | 0.279 | 28/51 (54.9) | 17/41 (41.5) | 0.216 |
Tumor grade III | 8/37 (21.6) | 15/63 (23.8) | 1.000 | 4/32 (12.5) | 19/68 (27.9) | 0.126 | 21/57 (36.8) | 2/43 (4.70) | <0.001 |
HER2-positive | 10/37 (27.0) | 13/63 (20.6) | 0.472 | 4/32 (12.5) | 19/68 (27.9) | 0.126 | 18/57 (31.6) | 5/43 (11.6) | 0.030 |
ER-positive | 18/37 (48.6) | 38/63 (60.3) | 0.300 | 25/32 (78.1) | 31/68 (45.6) | 0.003 | 21/57 (36.8) | 35/43 (81.4) | <0.001 |
Triple-negative |
8/37 (21.6) | 13/63 (20.6) | 1.000 | 4/32 (12.5) | 17/68 (25.0) | 0.193 | 18/57 (31.6) | 3/43 (7.0) | 0.003 |
Evaluated by Fisher’s exact test.
FOXP+, high infiltrate; FOXP3−, absent-low infiltrate of FOXP3-expressing lymphocytes,
ER−/PgR−/HER2− phenotype. LN, lymph node; FOXP3, forkhead box protein 3.
Univariate and multivariate analyses (Cox regression) for overall survival.
A, Univariate analysis.
| |||
Variable | HR | 95% CI | P-value |
| |||
Age (>50 years) | 0.61 | 0.23–1.61 | 0.318 |
Tumor size (>2 cm) | 2.06 | 0.78–5.41 | 0.135 |
LN metastasis | 1.74 | 0.63–4.78 | 0.279 |
Tumor grade III | 3.05 | 0.94–9.36 | 0.040 |
ER-positive | 0.38 | 0.14–1.04 | 0.050 |
HER2-positive | 2.35 | 0.89–6.17 | 0.075 |
Triple-negative |
1.39 | 0.45–4.27 | 0.565 |
Tumor FOXP3+, |
1.19 | 0.45–3.13 | 0.722 |
Cytoplasmic | 2.47 | 0.94–6.50 | 0.058 |
Nuclear | 0.13 | 0.02–0.95 | 0.016 |
Lymphocyte FOXP3+, |
5.87 | 1.34–25.69 | 0.008 |
Intensity of TILs | |||
High vs. absent-low | 0.71 | 0.14–3.68 | 0.685 |
Intermediate vs. absent-low | 1.01 | 0.35–2.96 | 0.984 |
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B, Multivariate analysis. | |||
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Variable | HR | 95% CI | P-value |
| |||
Tumor cytoplasmic FOXP3+ | 2.68 | 0.90–7.97 | 0.077 |
Tumor size (>2 cm) | 1.66 | 0.57–4.84 | 0.355 |
LN metastasis | 1.20 | 0.41–3.47 | 0.739 |
Tumor nuclear FOXP3+ | 0.15 | 0.02–1.16 | 0.070 |
ER-positive | 0.51 | 0.19–1.39 | 0.185 |
Lymphocyte FOXP3+, |
4.96 | 1.07–23.06 | 0.041 |
Tumor grade III | 0.88 | 0.27–2.92 | 0.836 |
ER-positive | 0.70 | 0.17–2.91 | 0.621 |
HER2-positive | 1.42 | 0.38–5.33 | 0.606 |
ER−/PgR−/HER2− phenotype;
cytoplasmic and/or nuclear FOXP3+;
high infiltrate of FOXP+ lymphocytes. HR, hazard ratio; CI, confidence interval; LN, lymph node; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; FOXP3, forkhead box protein 3; TIL, tumor-infiltrating lymphocyte.