A total of 52 patients who underwent primary systemic chemotherapy followed by definitive surgery during December 2005 and April 2008 at the Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital (Tokyo, Japan) were consecutively enrolled in this study. Informed consent was obtained from all patients and approval of the Institutional Review Board of Komagome Hospital was also obtained. All patients were diagnosed with invasive breast carcinoma by core needle biopsy and 4 patients were excluded since they were treated with primary hormonal therapy. Thus, 48 of the originally enrolled patients were evaluated. Four cycles of FEC (fluorouracil 500 mg/m², epirubicin 100 mg/m² and cyclophosphamide 500 mg/m²) administered intravenously (i.v.) on day 1 every 21 days were followed by four cycles of docetaxel i.v. (75 mg/m²) every 21 days, prior to surgery. None of the patients were administered with trastsuzumab prior to surgery.

The Japanese Pathological Response Criteria were applied, defined as follows: grade 0, no chemotherapeutic change in remnant cancer cells; grade 1a, 0–1/3 of remnant cancer cells in degeneration or necrosis; grade 1b, 1/3–2/3; grade 2, >2/3; grade 3, no viable cancer cells in duct and stroma (18,19).

Immunohistochemistry was performed to visualize the signal. Paraffin sections (4 μm) mounted on organosilane-coated glass slides were dewaxed in xylene and rehydrated through a graded ethanol series. The tissue sections were treated with a microwave antigen retrieval procedure in sodium citrate buffer (pH 6.0) for 20 min. They were subsequently treated with 0.3% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase activity. The primary antibodies were anti-DBC1 rabbit polyclonal antibody (produced in our laboratory) (8,9) and anti-SIRT1 rabbit polyclonal antibodies (H-300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). These primary antibodies were diluted (DBC1 1/100,000; SIRT1 1/200), and the tissue sections were incubated for 30 min at room temperature using reagents provided with the ChemMate EnVision™ Detection system (Dako, Carpinteria, CA, USA). Cells were visualized using the chromogen diaminobenzidine and counterstained with Mayer’s hematoxylin. Appropriate positive and negative controls were included.

For semi-quantitative evaluation of DBC1/SIRT1 expression, immunohistochemical scoring was performed, and nuclear staining of DBC1 and SIRT1 was evaluated according to the Allred score (20). The immunostaining results were interpreted as positive when at least 5% of cells were stained. No expression or expression of <5% of tumor cells was considered negative. The semi-quantification for immunostaining intensity was scored on a scale of: 0, negative; 1, weak; 2, moderate and 3, intense. Average numbers of immunopositive cells within the neoplastic tissues were determined in at least five areas at x400 magnification. The semi-quantification of the percentage of immunopositive cells was scored on a scale of 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%) and 4 (>75%). The percentage of the staining intensity and positive tumor cells were multiplied to produce a weighted score for each case. These scores were determined as the positive index. Immunohistochemical analysis was performed by three authors by consensus without knowledge of the clinicopathological information.

For ER, PR and HER2 evaluation, immunohistochemical staining was performed using anti-ER mouse monoclonal antibody (clone 1D5; Dako), anti-PR mouse monoclonal antibody (clone PgR636; Dako) and a HercepTest kit (Dako), respectively. Hormone receptor status was evaluated as the percentage of positive nuclear staining among cancer cells, and the cut-off value was set to 10%. HER2 scoring was carried out according to the standard HercepTest guidelines. HER2 expression status was further confirmed by fluorescence in situ hybridization using Vysis Path Vision HER2/neu DNA probe kit (Abott, Chicago, IL, USA), when the tumor was evaluated as 2⁺ by the HercepTest. The breast tumor samples were classified into four subtypes, namely luminal A (ER⁺ and/or PR⁺, and HER2⁻), luminal B (ER⁺ and/or PR⁺, and HER2⁺), HER2⁺ (ER⁻ and PR⁻, and HER2⁺) and triple-negative (ER⁻, PR⁻ and HER2⁻), according to the system for the immunohistochemical subtyping of breast cancer (21).

The association between DBC1 and SIRT1 expression and clinicopathological features was examined by the Mann-Whitney U-test. The correlation test was used to analyze correlations between SIRT1 and DBC1 using Spearman’s rank correlation test. All tests were two-tailed, and a P-value <0.05 was considered statistically significant.

The clinical characteristics and pathological data of the 48 breast cancer patients are shown in Table I. All patients were female and the median age of the enrolled patients was 53 years (range 32–75). Lymph node involvement was found in 18 patients and metastasis occurred in 22 patients. The TNM staging of the tumors ranged from stage 0 to stage IV: stage 0 (n=1), stage I (n=5), stage II (n=31), stage III (n=6) and stage IV (n=5). All tumors were graded according to the modified Bloom-Richardson system (22): grade 1 (n=12), grade 2 (n=10) and grade 3 (n=26). ER, PR and HER2 were positive in 28, 16 and 35 patients, respectively. The breast cancer subtypes in the present study included luminal A (n=8, 16.7%), luminal B (n=20, 41.7%), HER2⁺ (n=5, 10.3%) and triple-negative (n=15, 31.3%) subtypes.

DBC1 and SIRT1 immunoreactivity was present in the nuclei of normal and tumor cells. Positive expression of DBC1 and SIRT1 was noted in 85% (41 of 48) and in 98% (47 of 48) of patients, respectively. Representative immunostained tissues are shown in Fig. 1. Positive indices of DBC1 and SIRT1 judged by immunohistochemistry were calculated, and the association between the positive indices and the clinicopathological characteristics of the patients was further investigated. Elevated DBC1 expression was significantly associated with nuclear grade as determined by the modified Bloom-Richardson system (P= 0.019). Expression of DBC1 was inversely correlated with the HER2 expression status (P=0.026), while other clinical factors exhibited no significant correlation with the DBC1-positive index. Expression of SIRT1 was also negatively correlated with the HER2 expression status (P=0.003). However, in contrast to other studies, SIRT1 expression showed no relation with hormone receptor status and luminal subtype. We also analyzed the correlation coefficient between DBC1 and SIRT1 in breast cancer tissue and a marginal correlation was detected between the expression of DBC1 and the expression of SIRT1 (P=0.047, r=0.34).

Three patients were diagnosed with a grade 0 pathological response, and 22, 10, 6 and 5 patients were diagnosed with grades 1a, 1b, 2 and 3, respectively. To investigate whether DBC1 and SIRT1 expression is associated with chemotherapeutic response of the patients, the correlation between immunohistochemical positive index and pathological response of the invasive component of the breast carcinoma was evaluated, as pathological response is proposed to provide accurate information for prognosis. Expression of DBC1 and SIRT1 judged by immunohistochemistry was compared to the pathological response of the patients. Patients who exhibited a favorable response to neoadjuvant chemotherapy showed lower positive indices for DBC1 and SIRT1 rather than those who exhibited an unfavorable response, but this inverse relationship was not statistically significant (Table II).