We retrospectively reviewed the records of 323 consecutive breast cancer patients who received NAC at the Jikei University Hospital between November 2005 and March 2012. This study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983, and was approved by the Ethics Committee of the Jikei University School of Medicine; patient consent was also obtained. We evaluated the age of patients, clinicopathological characteristics such as clinical tumor size and clinical lymph node status before NAC, ER, PgR, and HER2 expression, pathological tumor size, pathological lymph node status, lymphovascular infiltration, epidermal growth factor receptor (EGFR), cytokeratin (CK) 5/6 as the basal marker, claudin-3 expression, and survival data for the patients. Lymphovascular infiltration was evaluated with the operative sample after NAC. A clinically node-positive axilla was defined as the presence of palpable mass in the nodal basin and, when assessed using ultrasound, magnetic resonance imaging, or computed tomography images, the presence of abnormal lymph nodes. When these had a suspected cancerous appearance on images, positivity was confirmed via fine needle aspiration.

All patients received NAC with four cycles of epirubicin (100 mg/m²), 5-fluorouracil (500 mg/m²), and cyclophosphamide (500 mg/m²), followed by four cycles of docetaxel (100 or 75 mg/m²). If patients were HER2-positive, trastuzumab was administered concurrently with docetaxel and adjuvant trastuzumab for one-year duration. A five-year adjuvant hormonal therapy was followed if the patients were hormone receptor positive. Patients who underwent breast conserving surgery received whole breast radiotherapy, and regional lymph node radiotherapy was used for patients with ≥4 -positive nodes. Post-mastectomy radiation therapy was administered to patients with initial tumors ≥5 cm or those with ≥4 positive nodes.

Systemic and breast examinations were performed before neoadjuvant chemotherapy, before surgery, and every 12 months postoperatively using chest and abdominal computed tomography, mammograms, breast ultrasonography, brain magnetic resonance imaging, and bone scans.

Immunohistochemistry (IHC) was evaluated using core needle samples before NAC and to ensure accuracy we used positive staining tissue as a control. IHC was performed according to the standard protocol on 3 µm sections of formalin-fixed paraffin embedded (FFPE) tissues using CONFIRM anti-ER rabbit monoclonal antibody (SP1; Roche Diagnostics Ltd.) for ER staining and CONFIRM anti-PGR rabbit monoclonal antibody (SP1; Roche Diagnostics Ltd.) for PgR staining. Nuclear staining ≥1% was considered positive. HER2 expression was determined using IHC with a rabbit polyclonal antibody (Agilent Technologies) on 4 µm sections of paraffin-embedded tissue. A staining score of 3⁺ according to the HercepTest criteria was considered positive; a result of 2⁺ was considered positive only if confirmed by fluorescence in situ hybridization with an amplification ratio of ≥2.0. Furthermore, on 3 µm FFPE tissue sections, the EGFR and CK5/6 expression were determined using IHC with mouse monoclonal antibodies (EGFR; dilution, 1:10; Leica Biosystems, cat. no. EGFR-L-CE, Wetzlar, CK5/6; dilution, 1:50; Agilent Technologies, cat. no. M7273). The expression of claudin-3 was determined using IHC with a rabbit polyclonal antibody (dilution, 1:100; Invitrogen; Thermo Fisher Scientific, Inc. cat. no. 18-7340) on 5 µm FFPE tissue sections. EGFR, CK5/6 and claudin-3 staining results were considered positive if any cytoplasmic and/or membranous invasive carcinoma cell staining was observed. If EGFR and/or CK5/6 were positive, the sample was considered basal marker positive.

pCR was defined as no evidence of residual tumor cells in the breast and in the lymph nodes.

We conducted Fisher's exact test to assess the association of clinicopathological characteristics and pCR between the patients with and without TNBC and that between basal marker and claudin expressions and pCR in the TNBC group. DFS was measured from the date of surgery to the date of any recurrence or the last follow-up. Overall survival (OS) was measured from the date of diagnosis to the date of death or the last follow-up. The Kaplan-Meier method was used to generate survival curves and the cumulative incidence of events. Cramér-von Mises test was used to assess the differences in Kaplan-Meier curves. The Cox regression model was used to identify the potential prognostic and predictive indicators. All significant tests were two-sided, a P-value ≤0.05 was considered statistically significant. All analyses were performed using Stata statistical software (Stata SE 10; StataCorp LP).

The median patients age was 53 years (range; 24-75 years). Table I shows the patient details and tumor characteristics. TNBC accounted for 20.4% of the total breast cancer. Age, clinical tumor size and node status before NAC, pathological tumor size and node status, and lymphovascular infiltration were not significantly different between TNBC and non-TNBC patients. Among the 323 patients, 26 patients (8%) achieved a pCR including 13 patients (19.7%) with TNBC and 13 (5.1%) without TNBC. The pCR rate of TNBC was significantly higher than that of the non-TNBC group (P<0.001). The reduction in tumor status was significant in patients with TNBC (P=0.001).

Core needle samples before NAC were available for basal marker and claudin staining for 43 patients with TNBC. We observed basal marker positivity in 25 (58.1%) cases of TNBCs and claudin-3 positivity in 21 (48%) cases with TNBC. Table II shows the associations between basal marker and claudin expressions and pCR. The pCR rate of basal marker-positive tumors was 24% and that of claudin-positive tumors was 25%. Basal marker-negative and claudin-negative tumors had the lowest pCR rate (0%) whereas basal marker-negative and claudin-positive tumors had the highest pCR rate (33.3%). The association between the basal marker and/or claudin expression and pCR rate were not statistically significant (P=0.134).

After a median follow-up time of 111.5 (range: 6.8-170.2) months, 23 patients showed local recurrence and 47 patients showed distant recurrence. Breast cancer related-disease occurred in 13 (19.7%) patients with TNBC (5-year DFS: 80.3%, 95% CI: 0.68-0.88; 10-year DFS: 80.3%, 95% CI: 0.68-0.88) and in 45 (17.5%) patients with non-TNBC (5-year DFS: 87.5%, 95% CI: 0.83-0.91; 10-year DFS 82.1%, 95% CI: 0.76-0.87). Fig. 1A shows the cumulative DFS by TNBC versus non-TNBC (Cramér-von Mises P<0.001). Overall, 8 (12.1%) patients with TNBC (5-year OS: 86.8%, 95% CI: 0.74-0.93; 10-year OS: 84.8%, 95% CI: 0.72-0.92) and 26 (10.1%) patients with non-TNBC died (5-year OS: 96.2%, 95% CI: 0.93-0.98; 10-year OS: 88.6%, 95% CI: 0.83-0.92). Fig. 1B shows the cumulative OS by TNBC versus non-TNBC (Cramér-von Mises P<0.001). Table III summarizes the results of univariate and multivariate analyses for survival. In the univariate analysis for DFS and OS, clinical tumor size, node status before NAC, pathological tumor size, pathological node status, and lymphovascular infiltration were statistically significant prognostic factors. In the multivariate analysis for DFS, clinical and pathological tumor size, and lymphovascular infiltration were independent prognostic factors. In the multivariate analysis for OS, pathological node-positive and lymphovascular infiltration were independent worse prognostic factors. Fig. 2 shows the cumulative survival of patients with TNBC. The patients with pathological node-negative status showed significantly good prognosis. (log rank P<0.001). Among TNBC patients with pathological node-positive status, negative lymphovascular infiltration was a significant favorable prognostic factor (Table IV).