Breast cancer is the most common malignant disease among women in the Western world and Japan. However, advances in the systemic treatment of breast cancer, particularly in chemotherapy, have contributed to declines in the breast cancer mortality rates (1). Anthracycline-containing regimens are the most widely used in the adjuvant and neoadjuvant settings for patients with breast cancer (2). Several previous clinical investigations have revealed that the use of neoadjuvant chemotherapy for patients with locally advanced breast cancer increased the surgical resectability rates and that the response to therapy correlated with the patients' ultimate disease-free survival (3–5). In addition, significant tumor volume reduction following neoadjuvant chemotherapy may permit subsequent breast-conserving surgical treatment (6,7) and a unique advantage of neoadjuvant chemotherapy is the possibility to take serial measurements of the primary tumor, therefore, allowing in vivo assessment of factors predictive of the sensitivity to the treatment (8).

Anthracyclines act via several mechanisms, however, the interaction with the nuclear enzyme topoisomerase IIα appears to be the most prominent mechanism (9). Topoisomerase IIα, which is a critical nuclear DNA binding enzyme, functions by reducing DNA twisting and supercoiling by cutting both strands of the DNA helix simultaneously, allowing selected regions of the DNA to untangle and to consequently engage in transcription, replication or repair processes. Disruption of topoisomerase IIα has been demonstrated to lead to double-stranded DNA breaks and cell death, and topoisomerase IIα is, therefore, also a proliferation marker of tumor cells, in addition to a target of anthracycline-based chemotherapy (10).

However, previous studies have reported variable expression levels of topoisomerase IIα and responses to anthracycline-containing chemotherapy in breast cancer, and while in vivo and in vitro studies each demonstrate that there is indeed an association between the expression levels of topoisomerase IIα and chemosensitivity to anthracyclines, these results remain controversial (11–14).

Gene expression profiling has identified distinct breast cancer molecular subtypes associated with different clinical outcomes. Breast cancer is a molecularly heterogeneous disease, which can be divided into ≥4 or 5 groups based on the expression profiles, including luminal A and B, normal breast-like, human epidermal growth factor receptor 2 (HER2)-positive, and basal-like (predominantly triple negative) breast cancer (15,16). Previous studies, including our previous study, revealed that triple negative breast cancer is associated with an improved pathological complete response rate compared with the other subtypes (17–19).

In addition, several biomarkers and intrinsic subtypes have been reported as predictors of the neoadjuvant response (20,21). However, no basis for selecting the optimal chemotherapy for individual patients has been determined, and the association between the expression of topoisomerase IIα and the different subtypes remains to be elucidated.

With this in mind, the present study aimed to retrospectively analyze whether the protein expression levels of topoisomerase IIα assisted in predicting the response to anthracycline-containing neoadjuvant chemotherapy among each breast cancer subtype and whether it is a prognostic marker of survival.

The present study used IHC to evaluate the expression levels of topoisomerase IIα, HER2, ER and PgR in tumor samples obtained from the pretreatment biopsies of breast cancer patients receiving an anthracycline-containing regimen as neoadjuvant chemotherapy. This retrospective data analysis suggested that the favorable response to anthracycline-containing neoadjuvant chemotherapy among the triple-negative and HER2-positive subtypes was independent of the expression of topoisomerase IIα.

Anthracyclines, including doxorubicin and epirubicin, which is less cardiotoxic compared with doxorubicin, are extensively used for the treatment of breast cancer, and anthracycline-containing polychemotherapy regimens have reduced breast cancer mortality by ~1/3 (1). The cardiac toxicity of anthracyclines is well described and the most common form, congestive heart failure, is known to be closely associated with the cumulative dose. Although limiting the cumulative dose to ~240–360 mg/m² doxorubicin has assisted in reducing the incidence of congestive heart failure to ~1.6–2.1%, data from long-term survivors of childhood cancer indicate that there is no true threshold for anthracycline-assoicated cardiotoxicity, and that cardiac damage may become apparent years later. However, studies from adjuvant breast cancer trials have shown that the likelihood of late cardiac effects in women who receive adjuvant anthracycline is low (2). However, since not all patients benefit from anthracyclines, a means of selecting the appropriate patients for the treatment is clearly of great interest. Anthracyclines have three major mechanisms of action: i) Inhibition of DNA and RNA synthesis by intercalating between the base pairs of the DNA/RNA strand; ii) enhancement of catalysis of oxidation-reduction reactions and iii) inhibition of topoisomerase IIα (9). Notably, the first mechanism also appears to be dependent on the inhibition of topoisomerase IIα for cytotoxicity.

Topoisomerase IIα is the only enzyme able to cleave and relegate double-stranded DNA. This enzyme acts during the relaxation of DNA supercoils, which accumulate during gene transcription and along with the progression of the replication fork. In addition, only topoisomerase IIα can perform the decatenation of replicated circular double-stranded DNA, and it is obligatorily involved in the remodeling of chromatin during mitosis. There are two highly homologous isoforms of topoisomerase II in humans, which are encoded by different genes. The gene for topoisomerase IIα is located on chromosome 17q21–22, while the gene for topoisomerase IIβ is located on chromosome 3q24 (10,23).

Drugs that interfere with topoisomerase IIα include anthracyclines (doxorubicin and epirubicin), etoposide, teniposide and amsacrine. These agents act by binding covalently with topoisomerase IIα following the occurrence of double-strand breaks, inducing lethal cellular damage by inhibition of relegation. An increase in the expression of topoisomerase IIα is associated with the sensitivity to these agents as a result of the increased substrate on which the drug may act.

Gene expression profiling has identified distinct breast cancer molecular subtypes (15,16) and previous studies have shown that triple-negative breast cancer is associated with an improved pathological complete response rate compared with the other subtypes (17–19). Nevertheless, the predictive role of topoisomerase IIα in each subtype remains to be elucidated. By contrast, HER2 amplification and overexpression have been reported as predictive markers of the benefit of anthracycline treatment in the adjuvant setting (14). Because of its location in the identical amplicon on chromosome 17, the gene encoding topoisomerase IIα (TOP2A) is frequently co-amplified with that of HER2 (24,25), which in turn leads to the overexpression of its protein product and possibly, to a greater sensitivity to anthracyclines (25–27). In 2011, Di Leo et al (14) performed a meta-analysis, in which they identified that HER2 amplification and TOP2A amplification and deletion may have certain value in the prediction of responsiveness to anthracycline-containing chemotherapy. However, non-HER2 amplified and non-TOP2A altered tumor types also appear to derive benefits from treatment with anthracyclines. Furthermore, in their meta-analysis, triple-negative breast cancer and moderately hormone-sensitive tumor types appeared to exhibit and improved response to anthracycline treatment compared with treatment with the cyclophosphamide, methotrexate and fluorouracil regimen. Therefore, a differential benefit from anthracyclines may exist within these subtypes. Since all triple-negative tumors, and ~90% of moderately hormone-sensitive tumors, from that previous study revealed no TOP2A gene amplification, other mechanisms of increased anthracycline sensitivity may exist. Du et al (13) suggested that topoisomerase IIα is a predictive factor for breast cancer patients who received anthracycline-containing neoadjuvant chemotherapy using fluorescence in situ hybridization in another meta-analysis. However, the authors could not detect an association between the expression of topoisomerase IIα and sensitivity to anthracycline-containing regimens using IHC, which is similar to the results of the present study.

Notably, the target of anthracycline is the topoisomerase IIα protein as opposed to the gene, and it is known that there is a lack of correlation between gene status and protein expression (28–30). Proliferation signals can lead to overexpression of the topoisomerase IIα protein independently of the TOP2 gene status (29,31). In normal cells, the expression of topoisomerase IIα is regulated according to the cell cycle. In proliferating cells, topoisomerase IIα becomes detectable in the late G1 phase, and the quantity gradually increases, peaking in G2/M. By contrast, increased expression of topoisomerase IIα is commonly observed in malignant tumors, irrespective of the cell cycle stage (10).

There are certain limitations to the present study. Triple-negative, moderately hormone-sensitive and HER2-positive tumors are characterized by high proliferation (15,32,33) and this data further confirmed that topoisomerase IIα overexpression was more frequently observed among the triple-negative and HER2-positive subtypes. However, ideally, quantification of nuclear concentrations of topoisomerase IIα protein may be a more appropriate way to investigate its predictive value as opposed to IHC alone. Furthermore, the present study included breast cancer patients treated with anthracycline combinations, as well as other drugs. Therefore, the use of these other drugs, including taxanes, cyclophosphamide and fluorouracil, may have influenced the activity of topoisomerase IIα and obscured any existing association.

In conclusion, the present findings do not justify the routine use of immunohistochemical staining of topoisomeras IIα as a predictive marker of the response to anthracycline-containing regimens. Women with triple-negative and HER2-positive tumors appear to derive benefits from anthracycline-containing chemotherapy independently of the expression of topoisomerase IIα.