Clinicopathological Significance of SMAD4 Expression in Breast Cancer

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Abstract

INTRODUCTION

Breast cancer is among the most common malignancies and a leading cause of death in women [1]. However, advances in surgical techniques and systemic therapies have improved the survival of breast cancer patients in recent decades [2]. Breast cancer is a heterogeneous disease with a variety of pathologic and molecular features. The prognosis of breast cancer and therapeutic decision-making are known to depend on classic immunohistochemistry (IHC) markers, namely estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Recently, genomic assays have been used to provide information and to help in decision-making for adjuvant treatment [3]. Differential expression of various proteins has been researched to develop more efficient options for diagnosis and treatment.

SMAD4 is a major downstream mediator in the signaling of the transforming growth factor beta (TGF-β) pathway. The TGF-β signaling process is initiated when TGF-β binds to the homodimers of the TGF-β type II receptor (TβRII) on the cell surface. TβRII recruits and activates the TGF-β type I receptor (TβRI). The activated TβRI phosphorylates SMAD2 or SMAD3, which heterodimerizes with SMAD4. These complexes translocate into the nucleus where they bind to DNA and regulate TGF-β dependent gene expression [4]. TGF-β is crucial in supporting tissue homeostasis through its ability to regulate cell proliferation, differentiation, migration, apoptosis, and development. The loss of SMAD4 expression results in the deprivation of a major factor of cell growth inhibition, contributing to carcinogenesis. SMAD4 has been previously identified as a possible tumor suppressor since SMAD4 mutations have been reported with high frequency in solid tumors [5,6]. The SMAD4 expression level has been reported to correlate with prognosis in many types of cancers, including colon [7], pancreatic [8], and esophageal [9] cancers. However, very few studies on SMAD4 expression and prognosis in breast cancer have been performed, and the results have been conflicting [10-13]. In the present study, we investigated SMAD4 expression using a tissue microarray (TMA)-based IHC assay and evaluated its clinicopathological and prognostic significance in breast cancer.

METHODS

IHC staining and interpretation of SMAD4 expression

Multiple 4-μm sections were cut using Leica microtome (Leica Biosystems, Wetzlar, Germany) and transferred to adhesive-coated slides. One section was routinely deparaffinized with standard xylene and hydrated using graded ethanol in water. It was stained with hematoxylin and eosin and covered with a coverslip. For IHC, the TMA slides were dewaxed by heating at 55°C for 30 minutes and by three 5-minute xylene washes.

Primary monoclonal mouse anti-Smad4 antibody (Santa Cruz Biotechnology, Santa Cruz, USA) was diluted (1:200) in goat serum and incubated at room temperature for 1 hour. After three 2-minute washes with phosphate-buffered saline, the sections were incubated with a biotinylated goat anti-mouse secondary antibody for 30 minutes (DAKO, Carpinteria, USA). The slides were then dehydrated following standard procedure and sealed with coverslips. Negative controls were performed by omitting Smad4 antibody during the primary antibody incubation.

To analyze SMAD4 expression in breast cancer, we used the IHC assay with the application of immunoreactivity score (IRS) criteria [16]. The extensional standards were the “fraction of positive cells” (0: <5%, 1: 6%−25%, 2: 26%−50%, 3: 51%−75%, and 4: >75%) and the “staining intensity score” (0: colorless, 1: pallide-flavens, 2: yellow, and 3: brown). The IRS was calculated by multiplying the “staining intensity score” and the “fraction of positive cells” [17]. The staining score was stratified as absent (−, score 0), weak positive (+, score 1−4), moderate positive (++, score 5−8), and strong positive (+++, score 9−12) (Figure 1). To evaluate SMAD4 expression, specimens with absent and weak positive (0 ≤IRS ≤4) scores were classified into the low SMAD4 expression (SMAD4 low) group. Specimens with moderate and strong positive scores (5 ≤IRS ≤12) were classified into the high SMAD4 expression (SMAD4 high) group (Figure 1). The interpretation of IHC staining in this study was performed by a single pathologist (SS Paik).

Figure 1.

Representative sections of the immunohistochemistry of SMAD4 in breast cancer tissue (magnification, × 200). (A) Negative SMAD4 staining. (B) Weak SMAD4 staining. (C) Intermediate SMAD4 staining. (D) Strong SMAD4 staining.

RESULTS

The median follow-up period was 66.3 months (range 3.9 to 134.2 months) and the median age was 49 years (range 27 to 79 years). Among the 255 cases, 239 (93.7%) patients had primary tumor size of less than 2 cm and 16 (6.3%) patients had primary tumor size of more than 2 cm. Regional lymph node metastasis was present in 131 cases (51.4%). According to the AJCC classification scheme, 77 (30.2%) patients had stage I cancer, while 178 (69.8%) had stage II and stage III cancer. ER expression was positive in 142 cases (55.7%) and PR expression was positive in 134 cases (52.5%). Out of all the cases, 67 (26.3%) were positive for HER2 expression on IHC analysis and/or positive for HER2 gene amplification detected by the FISH analysis. One hundred sixty-six specimens (65.1%) were categorized into the SMAD4 low group and 89 specimens (34.9%) were categorized into the SMAD4 high group (Table 1, Figure 1).

Table 1.

Association between SMAD4 expression and clinicopathological parameters in breast cancer patients (n=255)

Comparison between survival outcome and SMAD4 expression

During the follow-up period, there were 64 (25.1%) recurrences among the 255 patients. Out of these, 47 cases were SMAD4 low and 17 were SMAD4 high (73.4% and 26.6%, respectively). In overall survival, 55 events (21.6%) occurred. Out of these, 42 patients were SMAD4 low and 13 were SMAD4 high (76.4% and 23.6%, respectively). No difference was observed in DFS between the SMAD4 low group and the SMAD4 high group (p=0.115), but the SMAD4 high group showed significantly better OS (p=0.045) than the SMAD4 low group (Figure 2A, 2B). In the subgroup analysis, high SMAD4 expression was correlated with better OS in T1 stage tumor (size ≤2 cm) (p=0.034). However, it did not show significant correlation with DFS (Figure 2C, 2D). In the multivariate analysis, a significant association was observed between OS and SMAD4. High SMAD4 expression was observed to be an independent prognostic factor for better OS among patients with T1 stage tumor in the multivariate analysis (hazard ratio: 0.459, p=0.024) (Table 2).

Figure 2.

Kaplan-Meier curves for disease-free survival (DFS) and overall survival (OS) for breast cancer according to SMAD4 expression. (A) All patients –DFS. (B) All patient – OS. (C) T1 staged tumor (≤2 cm) – DFS. (D) T1 staged tumor (≤2 cm) – OS.

Table 2.

Univariate and multivariate analysis of various prognostic parameters for survival in cancer patients with primary tumor size ≤ 2 cm

DISCUSSION

SMAD4 is a key mediator of the TGF-β pathway. In the present study, the expression of SMAD4 was investigated using IHC analysis in 255 cases of IDC. The present data showed that high SMAD4 expression was associated with favorable clinicopathological parameters (stage, ER positivity, and HER2 negativity) and OS. There was a significant association between SMAD4 expression and OS in patients with T1 stage tumors (tumor size ≤2 cm).

SMAD4 expression was found to be lower in breast tumor cells when compared with normal breast epithelium [11]. Two previous reports applied the IRS criteria with respect to the expression of SMAD4 in breast cancer. Lui et al. [13] reported that 43.1% of breast cancer cases were SMAD4 low (0 ≤IRS ≤4) and 56.9% were SMAD4 high (5 ≤IRS ≤2 ), whereas Stuelten et al. [11] reported the figures of 36.3% and 64%, respectively. Our results are closely analogous to those of these studies, with 34.9% SMAD4 low cases and 65.1% SMAD4 high cases. There are inconsistent reports on the correlation between SMAD4 expression and prognostic markers in breast cancer. In previous studies, SMAD4 showed no significant correlation with tumor size, metastases, nodal status, histological grade, histological type, or estrogen receptor expression [11]. However, Lui et al. [13] reported that SMAD4 expression was negatively associated with histological grade.

The role of SMAD4 as a tumor suppressor is consistent with the observation that high expression of this protein is associated with a favorable prognosis. Recent studies have indicated that inactivation of SMAD4 is related to progression of disease in various cancers [18-21]. In colon cancer, SMAD4 inactivation promotes malignancy and drug resistance [22]. In pancreatic cancer, low expression of SMAD4 is associated with malignant progression [23]. Similarly, in non-small-cell lung carcinoma, SMAD4 expression was higher in the normal broncho-tracheal epithelium, but lower in the tumor tissues and closely correlated with lymph node metastasis [24]. Thus, low expression of SMAD4 shows an unfavorable outcome in other types of cancers. Our study demonstrated similar results in breast cancer.

Very few studies have reported the correlation between SMAD4 and prognosis in breast cancer. Lui et al. [13] reported that SMAD4 expression appeared to be decreased in breast cancer when compared with normal tissues and reduced SMAD4 expression levels tended to exhibit more poorly differentiated tumors, a higher risk of recurrence, and shorter OS. Kruijt et al. [12] also reported that low expression of SMAD4 had an unfavorable prognosis regarding progression-free survival. These results are in agreement with the present study. However, one study suggested that a SMAD4-negative tumor showed marginally better overall 5-year survival than a SMAD4-positive tumor, though this difference was not statistically significant [11]. Another study demonstrated that loss of SMAD4 was correlated with a decrease in axillary lymph node metastasis [25]. Since there are conflicting reports in terms of prognosis, recent studies have attempted to determine whether SMAD4 as a prognostic factor combines with other genes or protein expressions [10,12].

TGF-β is considered to play a dual role in cancer development as it displays both tumorigenic and tumor-suppressive effects. TGF-β has been reported to act as a tumor suppressor by inhibiting the cell proliferation of breast cancer cell lines in early stage [26,27]. In contrast, in later stages of cancer, TGF-β has direct pro-tumorigenic effects through the stimulation of invasion, the migration of tumor cells, and the activation of the tumor stroma [28,29]. Positive association of high expression of SMAD4 in T1 stage early breast cancer with better survival outcome in the present study could be explained by the fact that SMAD4 is a key mediator of TGF-β pathway and TGF-β acts as a tumor suppressor in the early stage.

The present study has several limitations. No stage IV patients were included in the TMA data. The IHC analysis using TMA may not reflect the intratumoral heterogeneity. Moreover, the proportion of small-sized tumors was unintentionally higher than the large-sized tumors in our data. However, our results could provide data for SMAD4 expression and prognosis in the early stage breast cancer.

In conclusion, high SMAD4 expression was correlated with several favorable prognostic factors including tumor size, regional lymph node metastasis, AJCC stage, ER positivity, and HER2 negativity. Additionally, high SMAD4 expression was associated with favorable OS in T1 stage breast cancer. Therefore, SMAD4 in breast cancer has potential prognostic significance and further investigation and understanding are needed to elucidate it.

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