In recent years, NAC has been adopted as one of the standard care regimens for primary resectable early-stage breast cancer. In such cases, the NAC generally consists of an anthracycline-containing regimen and taxane [4, 5]. The evaluation of the tumor response to NAC is important to determine the appropriate post-operative chemotherapeutic regimen for patients with recurrent tumors. There are various systems for classifying the survival response and pathological response in neoadjuvant trials—i.e., the cTMN, Fisher’s, Chevailler’s, and JBCS systems—and all of these have been shown to yield basically comparable results . In this study, we used the WHO and JBCS classifications as the therapeutic response criteria. The pCR rate was 29% (30/102), and the response rate was 78% (80/102). These response rates were similar to those previously reported [6, 9, 10].
The correlation between chemosensitivity and survival remains controversial. Some papers have reported that the prognostic factors included the clinical and pathological response to primary chemotherapy. On the other hand, at least one paper has reported that response classifications were inadequate as prognostic markers of the long-term outcome after NAC . Our data indicated that clinical response was not a significant predictor of DFS. Although clinical examination provides approximate indicators of chemotherapy responses, histopathologic examination of specimens after chemotherapy is important to evaluate the accurate response or the prognosis [40–43]. A tumor diagnosed as showing a complete clinical response sometimes retains residual carcinoma cells by microscopic examination; conversely, a palpable residual mass may show fibrosis without cancer cells [42, 43]. These findings might explain why the association between clinical response and DFS was more less statistically significant than that between pathological response and DFS in our study.
Our data indicated that pathological response was a ignificant predictor of the DFS. In this study, FEC followed by wPTX was the only NAC regimen used for patients with resectable early-stage breast cancer. However, the variety of chemotherapy regimens used as NAC in previous reports might have been a factor in producing these inconsistent results.
CA9, a hypoxia-associated endogenous protein, has been implicated in the regulation of the hypoxic microenvironment [44, 45]. CA9 is considered to be one of the cellular biomarkers of hypoxic regions in solid tumors. In the present analysis, CA9 was positive in CNB specimens from 47 (46%) of 102 patients, similar to the ratio in a previous study . CA9 expression was significantly associated with lymph node status and lymph-vascular invasion. CA9 has been shown to maintain the survival of breast tumor cells under hypoxic conditions . Breast cancer cells under hypoxic conditions might be associated with aggressive tumor phenotypes, which may indicate a poor prognosis for patients with CA9-positive breast cancer, as suggested in previous studies [29, 30, 48–50].
Tan et al. reported that CA9 in basal-like breast tumors was associated with resistance to chemotherapy (cyclophosphamide, methotrexate and 5-fluorouracil (CMF) or adriamycin and cyclophosphamide (AC)) and poor prognosis . In our present analysis of 31 triple-negative breast cancers, the DFS of patients with CA9-positive tumors was significantly shorter (p = 0.015) than that of patients with CA9-negative tumors (Additional file 2: Figure S1). The chemosensitivity of triple-negative breast cancer patients with CA9 was significantly lower than that of the CA9-negative cases (Additional file 1: Table S2). CA9 might be a useful biomarker for chemotherapy in triple-negative breast cancer. Supuran and colleagues found that selective CA9 inhibitors inhibited cell migration and spreading of breast cancer cells in the absence of oxygen, suggesting that CA9 is a pivotal target for antitumor therapy in patients with breast carcinoma [25, 51]. These findings suggest that CA9 inhibitors followed by wPTX chemotherapy might be useful in cases of breast carcinoma with resistance to FEC.
Biological markers predicting chemosensitivity have been evaluated in several studies, but there is still no clinically useful marker. ER- or PR-positive patients showed lower pCR rates after NAC than ER- or PR-negative patients. The pCR rate of CA9-positive tumors in CNB specimens was significantly lower than that of CA9-negative tumors. Multivariate analysis revealed that CA9 expression before NAC was an independent predictive factor for pCR. An extensive hypoxic microenvironment as determined by CA9 expression in breast cancer might play a significant role in the resistance to chemotherapy. These results may indicate that CA9 expression in CNB specimens is a useful marker for predicting chemosensitivity to NAC.
We also examined the correlation of CA9 expression between CNB tissues and resected tissues in the 72 patients. Although no significant correlation of CA9 staining was observed between the two groups, CA9 expression in resected tissues showed a tendency (p = 0.081) toward association with that in CNB tissues (Additional file 1: Table S3). CA9 expression after NAC (67%) was higher than that before NAC (46%). CA9-positive cells were observed more frequently in tumor specimens than in CNB specimens. Eleven of 32 patients with CA9-negative tumors before NAC were found to have CA9-positive tumors after NAC. NAC was thus effective in reducing CA9-negative cells, and resulted in an increase in hypoxic CA9-positive cells. Twenty-three of 55 patients with CA9-negative tumors before NAC achieved pCR, and could not be enrolled in the CA9 expression analysis because there was no tumor involvement detected in the resected tissues. These changes in CA9 expression before and after NAC might be one of the reasons for the lack of a significant correlation between the CA9 expression in CNB tissues and that in resected tissues.
CA9 expression in resected tissues after NAC was correlated with both prognosis and recurrence. In addition, multivariate regression analyses indicated that the CA9 expression level after NAC was an independent prognostic factor for DFS. Thus, CA9 expression after NAC may be a clinically informative prognostic marker for breast cancer patients treated with NAC. On the other hand, CA9 expression before NAC in CNB specimens may be a useful surrogate marker for predicting chemosensitivity. Our results indicate that the hypoxic marker CA9 in CNB specimens could be used to predict chemosensitivity, and that high expression of CA9 in resected tissue is correlated with worse outcomes in patients treated with FEC followed by wPTX chemotherapy.