The study cohort consisted of 198 TNBC patients, with a median age of 42 years (range, 24-71 years), who received NAC between January 2003 and September 2012, had known pathologic and clinical tumor responses, and from whom biopsy material was available for pathological evaluation. pCR was defined as the complete disappearance of invasive carcinoma cells in the breast and axillary lymph nodes after NAC (ypT0/is, ypN0). Patients with residual ductal carcinoma in situ were considered to have achieved pCR.

Eighty-two patients were treated with docetaxel (75 mg/m² or 60 mg/m²) and doxorubicin (60 mg/m² or 50 mg/m²) by intravenous infusion every 3 weeks for three cycles, followed by primary surgery and an additional three cycles of docetaxel and doxorubicin, and, if indicated, radiation therapy. An add-itional 74 patients received six cycles of neoadjuvant docetaxel and doxorubicin. Another 42 patients received four cycles of AC (doxorubicin 60 mg/m² or 50 mg/m², cyclophosphamide 500 mg/m² on day 1, every 21 days), followed by four cycles of docetaxel (75 mg/m² or 60 mg/m²). Four patients who showed progressive disease during NAC received adjuvant chemotherapy, consisting of FAC (fluorouracil, doxorubicin, and cyclophosphamide) or CMF (cyclophosphamide, methotrexate, and fluorouracil) after curative surgery. Patients underwent either mastectomy or breast-conserving surgery at the discretion of the surgeon. Sentinel lymph node biopsies were performed unless contraindicated. If radiotherapy was indicated, it was administered after surgery.

This retrospective study protocol was reviewed and approved by the Institutional Review Board (approval number: 1003-020-311) of the Seoul National University Hospital. Because this study was performed using a total of 198 consecutive patients in our database, and involved no more than minimal risk for the subjects, the Institutional Review Board approved our request for the waiver of informed consent. This study followed the recommendations of the Declaration of Helsinki for biomedical research involving human subjects.

Pretreatment biopsy material was used for immunohistochemistry (IHC) analyses to avoid any effects of chemotherapy on the expression levels of the proteins. Pretreatment formalin-fixed, paraffin-embedded tissue blocks were used for IHC. Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), p53, Bcl-2, and Ki-67 expression levels were evaluated by the avidin-biotin complex technique for IHC [6]. Tissue sections (4 µm thick) were cut from paraffin-embedded tissue blocks, deparaffinized in xylene, rehydrated with graded ethanol, and immersed in Tris-buffered saline. After antigen retrieval, the sections were incubated with primary antibodies as described [9]. Mouse monoclonal primary antibodies to ER (1:50), PR (1:50), p53 (1:1,200), Bcl-2 (1:50), and Ki-67 (1:800) were obtained from Dako Corp. (Carpinteria, USA), whereas antibody to HER2 (1:200) was obtained from Novocastra Laboratories Ltd. (New-castle-Upon-Tyne, UK). Sections were subsequently incubated with biotinylated antimouse IgG antibody and streptavidin horseradish peroxidase (Zymed Laboratories, San Francisco, USA). High expression of Ki-67 was defined as expression in ≥10% of tumor tissues, as the 10% cutoff provided the best predictive value for prognosis in our institution [10]. Bcl-2 expression was judged positive when ≥10% of the nuclei and cytoplasm of tumor cells were stained. Pathological records in our institution are designated as either positive or negative for Bcl-2 expression. IHC positivity in ≥25% of the tumor cells was considered high expression. This value was almost equivalent to the cutoff provided by a previous study in our institution [1]. Tumors were regarded as negative for ER and PR expression when <1% of the tumor cells showed nuclear staining. HER2 was considered negative when the HER2 score was 0 or 1+ upon IHC, or when the HER2 score was 2+ and HER2/CEP17 gene amplification was negative (as assessed by using fluorescence in situ hybridization). TNBCs were defined as ER-negative, PR-negative, and HER2-negative breast cancers.

The associations between the clinicopathological characteristics and pathological response after NAC were calculated using the chi-squared and Fisher exact tests. We used a stepwise regression model to approximate the risk ratio of achieving pCR in multiple regression analyses according to the baseline factors. Disease-free survival (DFS) was defined as the interval from the date of operation to the date when disease relapse or progression was first documented. Kaplan-Meier curves were generated to evaluate the DFS. All values were two-sided, and statistical significance was defined as p<0.05. SPSS version 19.0 software (IBM Corp., Armonk, USA) was used for all statistical analyses.

Of the 198 patients, 123 (62.1%) presented with clinical stage III at the time of initial diagnosis and 63 (31.8%) presented with clinical stage IIB. Median tumor size was 4.3 cm (range, 0.8-9.8 cm), and 132 women (66.7%) underwent breast-conserving surgery. The relationships between pretreatment clinicopathological characteristics and expression levels of p53, Ki-67, and Bcl-2 are shown in Table 1. None of these clinicopathological characteristics showed significantly different distributions according to the levels of these three biomarkers.

After NAC, the complete absence of residual invasive carcinoma cells in the breast was confirmed by histological examination in 42 of the 198 patients (21.2%); however, five of these 42 patients had residual carcinoma cells in the resected lymph nodes. Therefore, 37 of the 198 patients (18.7%) achieved pCR. Of the clinicopathological factors assessed, the initial tumor size was found to be significantly associated with pCR, whereas overexpression of p53 and Ki-67 (Table 2, Figure 1), and high nuclear grade indicated a high likelihood of achieving pCR (Table 2). The pCR rate was significantly higher in patients with high p53 expression (≥25%) than in those with low p53 expression (<25%) (27.1% vs. 6.8%, p<0.001), and it was almost significantly higher in patients with high Ki-67 expression (≥10%) than in those with low Ki-67 expression (<10%) (19.5% vs. 5.9%, p=0.056). In contrast, the level of expression of Bcl-2 was not significantly associated with the pCR rate. The sensitivity, specificity, positive predictive value, and negative predictive value of p53 expression for predicting pCR were 77.8%, 50.3%, 26.2%, and 90.9%, respectively. When p53 and Ki-67 were analyzed in conjunction, the pCR rate was the lowest (5.2%) in patients with low levels of both biomarkers, and it was the highest (25.8%) in patients with high levels of both (Figure 2). Considering the independent significant factors and the clinically important chemotherapy regimens, multiple regression analysis showed that p53 expression was the only independent factor associated with pCR (odds ratio, 3.961; p=0.003) (Table 3). The group that achieved pCR tended to show superior DFS than the group that failed to achieve pCR (Figure 3A). However, expression of p53 alone could not predict pCR prognosis accurately (Figure 3B).