Forkhead box M1 (FOXM1) expression predicts disease free survival and may mediate resistance to chemotherapy and hormonotherapy in male breast cancer

Abstract

BACKGROUND:

Male breast cancer (MBC) is a rare and neglected disease. Prognostic and predictive factors in MBC are extrapoled from trials conducted on its female counterpart.

OBJECTIVE:

Since the relationship between the transcription factor Forkhead box M1 (FOXM1) expression and the clinical response to chemotherapy and hormonotherapy in MBC remains unknown, we sought to investigate the predictive value of FOXM1 in MBC.

Methods:

FOXM1 expression was assessed in 130 MBC cases. Clinical significance was analyzed by Kaplan Meier curves, log-rank test and multivariate Cox regression analyses.

RESULTS:

Patients with high FOXM1 expression had a significantly lower response rate to chemotherapy (P = 0.045) and hormonotherapy (P = 0.029) than those with low FOXM1 expression. Multivariate analyses indicated that FOXM1 was an independent prognostic factor for disease free survival in MBC patients (P < 0.001).

CONCLUSIONS:

FOXM1 may have a reliable predictive significance in male breast cancer and thus may become an important target for male breast cancer therapy in the near future.

Introduction

Male breast cancer is a rare disease that represents less than 1% of all breast cancers [1,2]. Recent, studies on male breast cancer revealed notable differences compared to its female counterpart on the genomic, transcriptomic and proteomic level [3–10]. These findings suggest that male breast cancer should be considered as an entirely separate disease that needs more comprehensive analyses in order to define an optimal clinical management for these patients. Although the biology of female breast cancer is now well defined, knowledge bases of MBC is still incomplete. Thus, the panel of predictive and prognostic factors for male patients with breast cancer remains limited.

The Forkhead box protein M1, FOXM1, belongs to the Forkhead superfamily of transcription factors [11,12]. FOXM1 plays an important role in cell proliferation and cell cycle progression [13]. Furthermore, many studies demonstrated that FOXM1 plays an important role in all cancer hallmarks [14,15] which explains the increasing number of studies indicating that FOXM1 is an important target for cancer therapy that offers promising prospects [16–19]. Moreover, increased expression of FOXM1 has been found in solid tumors including male breast cancer [20] and it was highly correlated with tumor progression, poor prognosis and treatment resistance [21].

However, to date, predictive value of FOXM1 expression in MBC remains unknown. As part of the Understanding Male Breast cancer study (NCT03240510) we sought to investigate the predictive value of FOXM1 expression in male breast cancer.

Materials and Methods

Patients and tissue samples

Between 2004 and 2013, 130 surgical specimens of confirmed male breast cancer were reviewed. Clinicopathological data and follow-up information were reviewed from Medical records. The study was approved by the institutional ethical committee.

Immunohistochemistry

Immunohistochemical assays were performed on 4  μm-thick sections from FFPE blocks. FOXM1 staining was done with the rabbit monoclonal antibody anti-FOXM1 (1:300; H-300, Santa Cruz Biotechnology Inc., USA) as previously described [20]. Staining intensity of FOXM1 was scored by 2 experienced pathologists (IB-AG) as follows: negative (0), weak intensity (1), moderate intensity (2), and strong intensity (3). The percentage of FOXM1 positive cells was scored as: negative (0), from 1% to 25% (1), from 26% to 50% (2), from 51% to 75% (3) and from 76% to 100% (4). The final FOXM1 immunohistochemical scoring was determined by multiplying the percentage score and intensity score. Tumors were divided into ‘Low FOXM1 expression group’ and ‘High FOXM1 expression group’ using Score 6 as a cutoff point [22].

Moreover, immunohistochemical analysis of ER, PR, HER2 and Ki-67 proliferation index were used for breast cancer intrinsic subtype’s definition according to the St. Gallen criteria [23].

Statistical analysis

All statistical analyses were performed using SPSS 20.0 (IBM) software package. Correlation of the expression of FOXM1 with response to treatment was performed using a χ² test. Overall survival (OS) was defined as time from diagnosis to death and disease free survival (DFS) was defined as time from diagnosis to metastasis or local recurrence. The Kaplan-Meier methods were used to make survival curves and the log rank test was used to perform survival analysis. Factors with significant prognostic value in the univariate Cox regression model were evaluated with the multivariate Cox regression model to explore the independent effects of survival. P value <0.05 was considered statistically significant.

Results

The median age at diagnosis was 66 years (range 30–96). Invasive ductal carcinoma was the predominant histological type representing 97% of the cases (n = 126). Tumor size ranged from 0.5 to 10 cm (median 2). 63.8% of our patients were grade 2 (n = 83) and 59.2% were N+ (n = 77). 45.4% of our patients were classified as luminal A-like (n = 59), 44.6% luminal B-like (n = 58), 5% HER2 enriched (n = 6) and 5% triple negative (n = 7). 93% (n = 121) of patients presented FOXM1 expression, however, only 37% were classified as high FOXM1 expression group (n = 48). In our series, all of our patients had surgery, 36% received adjuvant radiotherapy (n = 48), 31.5% received adjuvant chemotherapy (n = 41), 4.6% received palliative chemotherapy (n = 6) and 36% received adjuvant hormonotherapy (Tamoxifen) (n = 47).

FOXM1 expression and disease free survival

The median follow-up was 12.5 months (range 1–132). In the overall population, the 5-year OS rate was 43% [95CI, 0.04–0.52]. The 5-year DFS rate was 75.5% for the Low FOXM1 expression group and 29% for the High FOXM1 expression group. Univariate analysis showed that high FOXM1 expression (P < 0.001) and age (P < 0.001) were significantly associated with DFS (Table 1). Moreover, multivariate analysis identified only FOXM1 expression as an independent predictive factor for DFS of male breast cancer patients (P < 0.001, HR = 4.6 [2.436–9.135]) (Table 1). Furthermore, Kaplan-Meier survival curves revealed that high FOXM1 expression was associated with shorter DFS in male breast cancer (Fig. 1).

FOXM1 expression and response to chemotherapy

In our study, 34.6% of patients (n = 45) progressed after cytotoxic chemotherapy. 60.4% of the High FOXM1 expression group (29/48) progressed after chemotherapy, whereas only 19.5% of the Low FOXM1 expression group (18/82) progressed after chemotherapy. This difference was statistically significant (P = 0.045) (Fig. 2). Median progression free survival under chemotherapy was 8.5 months (range 1–132) for the High FOXM1 expression group and 15 months (range 1–50) for the Low FOXM1 expression group.

FOXM1 expression and response to hormonotherapy

In our study, 25.4% of patients (n = 33) progressed after Tamoxifen. 46% of the High FOXM1 expression group (22/48) progressed after Tamoxifen, whereas only 13.5% of the Low FOXM1 expression group (11/82) progressed after Tamoxifen. This difference was statistically significant (P = 0.029) (Fig. 3). Median progression free survival under Tamoxifen was 7 months (range 1–21) for the High FOXM1 expression group and 12 months (range 1–50) for the Low FOXM1 expression group.

Discussion

Male breast cancer is an uncommon disease that for a long time thought to be identical to female breast cancer. Lately, efforts towards better understanding the biology of male breast cancer are increasing, they outlined the differences hidden behind the similarities between male and female breast cancer [7–10]. Thus, management of male breast cancer should not be extrapolate from research carried out on female patients with breast cancer. Since male breast cancer has a limited biomarker panels, we assessed FOXM1 as a potential predictive biomarker for male breast cancer.

FOXM1 is intimately involved in tumorigenesis since it contributes to tumor initiation and progression, angiogenesis as well as epithelial-mesenchymal transition (EMT) [24–28]. FOXM1 is overexpressed in a wide range of cancers including male breast cancer [20]. Several studies reported that high FOXM1 expression is associated with poor prognosis [29]. In fact, high FOXM1 expression correlates with decreased overall survival, and disease free survival, increased incidence of lymph node metastasis, advanced stage and higher histological grade [21,30,31]. Moreover, we recently demonstrated that FOXM1 is also associated with poor prognosis in male breast cancer [20].

Drug resistance, either acquired or inherent, constitutes a major clinical challenge. The mechanisms underlying drug resistance are multiple and complex, and not fully understood. Therefore, identifying predictive factors to drug resistance is warranted in order to insure an optimal therapeutic management. Numerous studies indicates that FOXM1 induces resistance to treatment [33,34]. Research conducted on cancer cells revealed that FOXM1 expression is correlated with resistance to paclitaxel, anthracycline and cisplatin [34,35]. Furthermore, resistance to hormonotherapy, Herceptin and lapatinib in breast cancer was observed in breast cancer cell lines with high expression of FOXM1 [36,37]. Taken together, these findings showcase the significant role played by FOXM1 in resistance to cancer therapy, and highlight the potential success of FOXM1 targeted therapy.

Despite the fact of the emerging evidence of FOXM1 importance as a predictive marker to drug resistance, the predictive impact of FOXM1 has not been analyzed for male breast cancer so far. We therefore assessed FOXM1 as a potential predictive marker in male breast cancer. In this report conducted on 130 male breast cancer cases, we found that High FOXM1 expression was significantly associated with chemotherapy and endocrine resistance. In fact, patients with high FOXM1 expression presented higher levels of progression under both chemotherapy and hormonotherapy. These results are aligned with literature findings in female breast cancer along with other cancer types [32,33]. Furthermore, FOXM1 expression was identified as an independent predictive factor for disease free survival in male breast cancer patients.

Although the prognostic value of FOXM1 has been established in numerous solid tumors including breast cancer in both genders [29], the use of FOXM1 as a predictive biomarker of response to FOXM1 targeting therapies needs to be clinically validated. In the breast cancer setting, whether it’s in male or female, once targeted therapy anti-FOXM1 is available, it should be evaluated through early pilot studies in different molecular subtypes especially in the most challenging breast cancer subtype triple negative breast cancer (TNBC). Since to date, the treatment of TNBC is essentially based on chemotherapy and lacks targeted therapies, FOXM1 inhibitors may represent new promising therapeutic option.

Our study is limited by the retrospective design; however, these findings demonstrate that FOXM1 may have a reliable predictive significance in male breast cancer and may promote drug resistance. Further explorations are warranted in order to confirm these results.

Conclusions

In recent years, many studies highlighted the predictive value of FOXM1 in a wide range of cancers including female breast cancer but to the best of our knowledge not in male breast cancer. In our study we demonstrated that FOXM1 is a potential novel predictive biomarker in male breast cancer.