Patients (n=628) with histologically confirmed breast cancer who underwent radical surgery between January 2001 and January 2010 in Harbin Medical University (Nangang, China) were enrolled in the present study. Samples were obtained for immunohistochemical staining as well as prognostic analysis. The mean age of the patients was 47.28±9.43 years (range, 27–78 years). Patients underwent curative surgery, the resected specimens were pathologically examined and >10 lymph nodes were pathologically examined following surgery. Complete medical records including the ER, PR, Her2, p53 and Ki67 status were available. The study protocol was approved by Harbin Medical University. Patients were informed of the details of the study and agreed to participate.

For western blot analysis, cells were lysed with buffer (0.1% SDS, 50 mmol/l Tris-HCl, pH 7.6; 1% NP-40, 150 mmol/l NaCl, 2 mg/ml aprotinin, 2 mg/ml leupeptin and 7 mg/ml PMSF). The protein concentrations were determined using the bicinchoninic acid Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins (30 µg) were separated on 10% SDS-PAGE gels (Varsal Instruments, Beijing, China) and transferred to a polyvinylidene difluoride membrane (Varsal Instruments). After blocking, the membrane was incubated with an anti-DEK antibody (cat. no. ab166624; 1:1,000; Abcam, Cambridge, MA, USA) at 4°C overnight. After washing, the membrane was incubated with a secondary antibody (cat. no. ZB-2301; Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) at a dilution 1:3,000 at room temperature for 1 h. Proteins were detected with an enhanced chemiluminescent kit (Varsal Instruments) and anti-β-actin antibody (cat. no. SAB5500001; 1:1,000; Sigma-Aldrich, St. Louis, MO, USA) was used as loading control. Densitometry was performed using Gel-pro Analyzer 4.0 (Media Cybernetics, Silver Spring, MD, USA).

Thin slices of tumor tissue from all cases were fixed in 4% formaldehyde solution (pH 7.0) for periods not exceeding 24 h. Paraffin embedding was conducted, and 4 µm-thick sections were cut and placed on glass slides coated with 3-aminopropyl triethoxysilane (Seebio Biotech Inc., Shanghai, China) for immunohistochemistry. Tissue samples were stained with hematoxylin and eosin to determine histological type and grade of tumors.

Briefly, breast tumor tissues were cut at a thickness of 4 µm using a cryostat. The sections were mounted on microscope slides, air-dried and then fixed in a mixture of 50% acetone and 50% methanol. The sections were then de-waxed with xylene, gradually hydrated with gradient alcohol, and washed with phosphate-buffered saline (PBS). Sections were incubated for 60 min with the primary antibody. Following washing with PBS, sections were incubated for 30 min with the secondary biotinylated antibody (Multilink Swine anti-goat/mouse/rabbit immunoglobulin; Dako Inc., Carpinteria, CA, USA). Following washing, Avidin Biotin Complex (1:1,000 dilution, Vector Laboratories Ltd., Burlingame, CA, USA) was then applied to the sections for 30–60 min at room temperature. The immunoreactive products were visualized by catalysis of 3, 3-diaminobenzidine (DAB) by horseradish peroxidase in the presence of H₂O₂, following extensive washings. Sections were then counterstained in Gill's hematoxylin and dehydrated in ascending grades of methanol prior to clearing in xylene, and mounting under a coverslip. The sections were observed under an Olympus CX31 microscope (Olympus, Tokyo, Japan).

To score DEK as immunopositive staining, the positive cells are shown as a yellow to brown color in the nucleus and/or cytoplasm. DEK expression was classified semi-quantitatively according to the following criteria: −, <1% of neoplastic cells discretely expressed DEK; +, ≥1 of morphologically unequivocal neoplastic cells discretely expressed DEK.

All data were analyzed with SPSS statistics software (Version 13.0, SPSS Inc., Chicago, IL, USA). Correlations between DEK and other parameters were investigated using the χ² test, Fisher's exact test or independent t-tests. The Kaplan-Meier method was adopted to analyze disease-specific survival, while the log-rank test was used to analyze survival differences. Multivariate analysis was performed using the Cox proportional hazards model selected in forward stepwise. P<0.05 was considered to indicate a statistically significant difference.

Immunohistochemical examination showed that DEK was located in the nucleus and/or cytoplasm of breast cancer cells. It was observed that expression of DEK protein was significantly higher in breast cancer tissues compared with paracancerous tissue (61.94% vs. 6.53%; Fig. 1). Western blot analysis showed that DEK protein was significantly highly expressed in breast cancer tissues with lymph node metastasis compared with those without (P=0.001; Fig. 2). After universal analysis, DEK was observed to be correlated with age, tumor size, histological type, lymph node metastasis and distant metastasis (P=0.024, 0.001, 0.001, 0.001 and 0.001, respectively; Table I).

Patients with high expression of DEK were shown to have a significantly increased distant metastasis rate. Furthermore, 185 (74.30%) of 249 breast cancers with distant metastasis exhibited DEK expression compared with 204 (53.83%) of 379 cases of non-distant metastasis (P=0.001).

The factors associated with post-operative distant metastasis with multiple analyses were also investigated. Age, tumor size, histological type, triple negative subtype and DEK expression were found to be associated with post-operative distant metastasis in breast cancer (Table II). In addition, the present study investigated the correlation between DEK expression and chemotherapeutic sensitivity in 107 patients with breast cancer who underwent neoadjuvant chemotherapy. DEK expression was expressed in 78.57, 67.86, 46.30 and 18.18% of patients with complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) (P=0.006; Table III).

Furthermore, DEK along with age, histological type, lymph node metastasis, affected survival rate in triple-negative breast cancer. Patients with triple-negative breast cancer and high DEK expression exhibited a poorer disease-specific survival compared with those with none or low expressed DEK protein (P=0.001; Fig. 3). In the Cox regression test, DEK protein was detected as an independent prognostic factor (P=0.001; Table IV).