Advantages of adjuvant chemotherapy for patients with triple-negative breast cancer at Stage II: usefulness of prognostic markers E-cadherin and Ki67

This study investigated a consecutive series of 1,063 cases of sporadic invasive breast carcinoma. Because E-cadherin is functionally silenced in invasive lobular carcinoma [34], 27 cases of invasive lobular carcinomas were excluded. Then a total of 1,036 breast cancer cases were enrolled in this study. All patients had received a curative operation of a mastectomy or a conservative surgery with axillary lymph node dissection in Osaka City University Hospital or Osaka City General Hospital from 2000 to 2006. The median follow-up time was 3.6 years (range, 0.2 to 6.0 years). Tumors were confirmed histopathologically and staged according to the TNM classification [35]. All patients who underwent breast-conserving surgery were administered post-operative radiotherapy. Adjuvant chemotherapy was performed either by an anthracycline-based regimen (doxorubicin or epirubicin) or by a 5FU-based regimen in TNBC depending on the stage or risk of recurrence in accordance with the National Comprehensive Cancer Network (NCCN) guidelines [36]. This study was conducted with the consent of the ethical committee of Osaka City University, and informed consent was obtained from all subjects. Overall survival time was set in days as the period from the initial surgery.

Immunohistochemical study was performed as previously reported [37]. Briefly, the formalin embedded tissue sections were deparaffinized, and were heated in Target Retrieval Solution (Dako, Carpinteria, CA, USA). Sections were then incubated in 10% normal goat or rabbit serum to reduce non-specific antibody binding. Tissue sections were then incubated with each primary monoclonal antibody against ER (clone 1D5, dilution 1:80; Dako, Cambridge, UK), PR (clone PgR636, dilution 1:100; Dako), HER2 (Hercep Test, Dako), p53 (clone DO-7, dilution 1:50; Dako), Ki67 (clone MIB-1, dilution 1:00; Dako) and E-cadherin (clone NCH-38, dilution 1:200; Dako). The slides were treated with streptavidin-peroxidase reagent, and were incubated in PBS diaminobenzidine and 1% hydrogen peroxide v/v, followed by counterstaining with Mayer's hematoxylin. Positive and negative controls for each marker were used according to the supplier's data sheet (Dako). Immunohistochemical scoring was performed in a blind fashion. The cut-off for ER positivity and PR positivity was > 0% positive tumor cells with nuclear staining. HER2 was graded according to the accepted grading scheme as 0, 1+, 2+, 3+. the following criteria were used for scoring: 0, no reactivity or membranous reactivity in less than 10% of cells; 1+, faint/barely perceptible membranous reactivity in 10% of cells or higher or reactivity in only part of the cell membrane; 2+, weak to moderate complete or basolateral membranous reactivity in 10% of tumor cells or higher; 3+, strong complete or basolateral membranous reactivity in 10% of tumor cells or higher. HER-2 was considered to be positive if immunostaining was 3+ or if a 2+ result showed gene amplication by fluorescent in situ hybridization (FISH). In FISH analyses, each copy of the HER2 gene and its centromere 17 (CEP17) reference were counted. The interpretation followed the criteria of the ASCO/CAP guidelines for HER2 IHC interpretation for breast cancer [38]: positive if the HER2/CEP17 ratio was higher than 2.2. The cut-off for p53 was ≥ 1% positive tumor cells with nuclear staining. Ki67-labelling index > 30% was determined to be positive. E-cadherin antibody intensely stained the membrane and weakly stained the cytoplasm of cancer cells. E-cadherin expression was semi-quantitatively analyzed according to the percentage of cells showing membrane positivity: 0, 0 to 10%; 1+, 10 to 30%; 2+, 30 to 70%; 3+, > 70%. E-cadherin expression was considered positive when scores were ≥ 2, and negative when scores were ≤ 1. Cytoplasmic staining only was not included in the assessment. E-cadherin antibody intensely stained the membrane and weakly stained the cytoplasm of cancer cells. A case with cytoplasmic staining only was determined as E-cadherin negative, as shown with "score 0" (Figure 1).

Cases that were negative for ER, PR and HER2 expression were considered to be cases of TNBC. Among the total 1,036 breast cancer cases, there were 190 (18.3%) cases of TNBC. Adjuvant chemotherapy was performed for 138 (72.6%) of the 190 TNBC cases; 60 cases had an anthracyclin-based regimen and 78 a 5FU-based regimen (Table 1). The remaining 52 cases received surgery alone. Among the 190 TNBC cases, those receiving surgery plus adjuvant therapy (n = 138) had a more favorable prognosis (P = 0.0043) than those undergoing surgery alone (n = 52) (Figure 2a). When restricting the analysis to patients with Stage II cancers, the overall survival of the surgery plus adjuvant chemotherapy group was significantly better than that of the surgery alone group (P = 0.0013) (Figure 2b). In contrast, in patients with Stage I and III cancers, no significant difference of overall survival was found between the surgery plus adjuvant chemotherapy group and the surgery alone group (Additional file 1). There was no significant difference in the prognosis of TNBC patients receiving adjuvant therapy between the anthracyclin-based regimen and 5FU-based regimen (Table 2).

Expression of E-cadherin, Ki67, and p53 was positive in 109 (57%), 65 (34%) and 118 (62%) of 190 cases of TNBC, respectively (Table 3). The Ki67 expression level was significantly high in Stage II and III TNBC tumors (63%, P = 0.013). No significant association between E-cadherin or p53 expression and clinicopathological parameters was identified in TNBC. The cases with E-cadherin-negative and Ki67-positive expression had a significantly higher incidence of lymph node metastasis (45%, P = 0.027).

TNBC with reduced E-cadherin expression showed a significantly worse overall survival time (P = 0.0054, log-rank), and cases with Ki67 expression showed significantly worse overall survival time (P = 0.0181, log-rank) (Figure 3a). Patients with E-cadherin-negative and Ki67-positive expression showed a significantly worse overall survival time (P = 0.001, log-rank). The prognosis of the E-cadherin-negative cancer patients was significantly poorer than that of the E-cadherin-positive cancer patients in regard to overall survival at Stage II (P = 0.0058) (Figure 3b). The prognosis of the combination of E-cadherin-negative and Ki67-positive expression cancer patients was significantly poorer than that of the combination of E-cadherin-positive and Ki67-negative expression cancer patients in regard to overall survival at Stage II (P = 0.0376) (Figure 3b) and Stage I (P = 0.0437) (Additional file 2). The combination of E-cadherin-negative and Ki67-positive expression was revealed to be a more significant prognostic indicator than either E-cadherin or Ki67 expression alone by multivariate analyses (P = 0.004, OR = 2.784), while a univariate analysis revealed that the overall survival was significantly correlated with the E-cadherin expression, Ki67 expression, stage, tumor size and lymph node metastasis (Table 2).

In the 138 TNBC cases undergoing surgery plus adjuvant chemotherapy, the prognosis of patients with E-cadherin-negative plus Ki67-positive expression was significantly worse that the prognosis of patients with either of these risk factors alone (P = 0.001), while no significant difference in prognosis was found in the 52 TNBC cases undergoing surgery alone (Figure 4a). No significant difference in prognosis was found between the E-cadherin-negative and Ki67-positive cases undergoing surgery plus adjuvant chemotherapy (n = 51) and surgery alone cases. A multivariate logistic regression analysis showed that the combination of E-cadherin-negative and Ki67-positive expression was significantly correlated with the overall survival of patients undergoing surgery plus adjuvant chemotherapy (P = 0.001), but not in those undergoing surgery alone, suggesting that E-cadherin-negative and Ki67-positive expression is an independent prognostic factor for TNBC with adjuvant chemotherapy (Table 4). In the adjuvant chemotherapy group, the overall survival of TNBC patients having both E-cadherin-negative and Ki67-positive expression was significantly worse than that of patients with either risk factor alone at each of Stages II (Figure 4), I and III (Additional file 3). In contrast, no significant difference in prognosis in relation to the Ki67 expression and/or E-cadherin expression was found in 52 cases without adjuvant chemotherapy at each of Stages II (Figure 4), I and III (Additional file 3).