A consecutive series of 266 patients with invasive ductal carcinoma were enrolled in this study. All cases were diagnosed and underwent surgery at the Hanyang University Hospital (Seoul, Korea) between August 2000 and January 2009. This study was approved by the Institutional Review Board of the Hanyang University Hospital (HYU 2014-11-005-002). The mean age of the patients was 50 years and the mean follow-up period was 60 months. Of the 266 cases, 42, 152, and 72 were histological grades 1, 2, and 3, respectively. According to the seventh edition of the American Joint Committee on Cancer (AJCC) staging system, 194 cases were stage I or II and 72 were stage III or IV. In addition, 16, 12, and 47 samples of normal breast tissue, usual ductal hyperplasia, and ductal carcinoma in situ, respectively, were randomly selected to evaluate the role of DUSP4 expression in carcinogenesis and tumor progression. We reviewed all hematoxylin and eosin (H&E)-stained slides, pathology reports, and other medical records to confirm the diagnosis. The pathological parameters assessed included age, tumor size, tumor grade, perinodal tumor extension, lymph node metastasis, estrogen receptor (ER) and progesterone receptor (PR) status, c-erbB-2 expression, and patient survival.

We used a manual tissue microarrayer (Unitama, Seoul, Korea) for tissue microarray construction from archival formalin-fixed, paraffin-embedded tissue blocks. As previously described [10], we selected areas rich in tumor cells by light microscopy of H&E-stained sections. Tissue cylinders of 2 mm diameter were punched from a previously marked lesion on each donor block and transferred to the recipient block (Unitama). Each tissue microarray was comprised of 5×10 samples.

Antibodies, polyclonal rabbit anti-DUSP4 (Abcam, Cambridge, UK), monoclonal mouse anti-ER (Novocastra Laboratories, Newcastle, UK), monoclonal mouse anti-PR (Novocastra Laboratories), and monoclonal mouse anti-c-erbB-2 (Novocastra Laboratories), were diluted 1:150, 1:50, 1:100, and 1:800 in goat serum, respectively. For immunohistochemical staining, 4-µm sections were cut from the tissue microarray block using a Leica microtome, transferred to adhesive-coated slides, and deparaffinized. The staining was performed using the Bond Max automated immunostainer (Vision Biosystems, San Francisco, USA). Before staining, heat-induced epitope retrieval was performed using the Bond epitope retrieval solution. We blocked endogenous peroxidase activity with 0.3% hydrogen peroxide. Slides were incubated in primary antibody for 30 minutes at room temperature and the slides were incubated with postprimary reagent for 15 minutes at room temperature. The reactions were developed using the Bond polymer refine detection kit and visualized with the chromogen, 3, 3'-diaminobenzidine tetrahydrochloride.

DUSP4 expression was evaluated semiquantitatively by two independent pathologists (H.K. and S.S.P.) who were blind to the patients' clinical outcome. We categorized cytoplasmic DUSP4 expression in terms of both staining intensity and extent, as described previously [11]. Staining intensity was graded as negative ( 0), weak ( 1), moderate ( 2), and strong ( 3), and staining extent was graded as 0% ( 0), 1%-25% ( 1), 26%-50% ( 2), 51%-75% ( 3), and 76%-100% ( 4). The product of intensity and extent grade was used as the final staining score. Thus, the maximum combined score was 12 and the minimum score was 0. Representative photomicrographs of DUSP4 immunostaining in invasive ductal carcinoma are shown in Figure 1. For the purpose of statistical analysis, a cutoff value of 4 was adopted according to the receiver operating characteristic curve. Therefore, the samples were finally classified as either negative (score 0-3) or positive (score 4-12) for DUSP4 expression. ER, PR, and c-erbB-2 expression status was interpreted according to the American Society of Clinical Oncology/College of American Pathologists guidelines for ER/PR/c-erbB-2 testing in breast cancer. When ≥1% of the tumor cell nuclei were stained, it was classified as ER or PR positive. Positive c-erbB-2 staining was determined by complete and intense membranous patterns in >10% of the tumor cells.

Statistical analysis was performed using the SPSS software version 19.0 (IBM Corp., Armonk, USA). The Mann-Whitney U test, chi-square test for linear trend, and chi-square test for independence were used to examine the association between DUSP4 expression and the clinicopathological parameters including age; histological grade; primary tumor (T) category; regional lymph nodes (N) category; AJCC stage; lymphatic invasion; perinodal tumor extension; expression of ER, PR, and c-erbB-2; and triple negativity. Spearman analysis was used to obtain the correlation coefficient. We used the Kaplan-Meier method with the log-rank test to perform analyses of overall and disease-free survival. To identify the independent prognostic factors, the Cox proportional hazards regression model was used in both univariable and multivariable analyses. A p-value <0.05 was considered significant.

In this study, we evaluated DUSP4 expression in 16, 12, 47, and 266 cases of normal breast tissue, usual ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma, respectively. Various grades of cytoplasmic DUSP4 expression were observed. DUSP4 expression was positive in 3 of 28 (10.7%) benign cases (normal breast tissue and usual ductal hyperplasia), 11 of 47 (23.4%) ductal carcinoma in situ cases, and 80 of 266 (30.1%) invasive ductal carcinoma cases (Table 1). DUSP4 was more frequently expressed in malignant than in benign cases (p=0.024). The mean DUSP4 expression score was 0.86±2.16 in normal breast tissue and usual ductal hyperplasia, 2.19±2.98 in ductal carcinoma in situ, and 2.40±3.30 in invasive ductal carcinoma (Figure 2). It was significantly higher in malignant tumors than in benign lesions (p=0.019, Kruskal-Wallis test).

We evaluated the correlation between DUSP4 expression and the clinicopathological parameters to assess the significance of its expression in invasive ductal carcinoma. DUSP4 expression was more frequent in the higher T-stage categories (T2 to T4) than in the lower one (T1) (Table 2), and significantly correlated with a larger tumor size (>2 cm, p=0.015). However, there was no correlation with histological grade, AJCC stage, lymphatic invasion, perinodal tumor extension, hormone receptor status, c-erbB-2 expression, or triple negativity.

The impact of DUSP4 expression on survival in 266 patients with invasive ductal carcinoma was evaluated. We observed that AJCC stage, lymphatic invasion, perinodal tumor extension, ER/PR status, and triple negativity showed a significant effect on overall and disease-free survival in univariable and/or multivariable analyses (Table 3). However, there was no significant correlation between overall survival or disease-free survival and DUSP4 expression (p=0.924 and p=0.246, respectively; log-rank test) according to the Kaplan-Meier survival curves for all 266 patients with invasive ductal carcinoma (Figure 3A, B). We evaluated the impact of DUSP4 expression on survival in 120 patients with T1-stage tumors (Table 4). We found that DUSP4 expression had a significant effect on overall and disease-free survival in multivariable analysis. Kaplan-Meier survival curves revealed a significant effect of DUSP4 expression on overall survival (p=0.034, log-rank test) and disease-free survival (p=0.045, log-rank test) in early T-stage breast cancer (Figure 3C, D).