Annexins in human breast cancer: Possible predictors of pathological response to neoadjuvant chemotherapy

https://doi.org/10.1016/j.ejca.2008.12.026Get rights and content

Abstract

Neoadjuvant chemotherapy is used in women who have large or locally advanced breast cancers. However, up to 70% of women who receive neoadjuvant chemotherapy fail to achieve a complete pathological response in their primary tumour (a surrogate marker of long-term survival). Five proteins, previously identified to be linked with chemoresistance in our in vitro experiments, were identified histochemically in pre-treatment core needle biopsies from 40 women with large or locally advanced breast cancers. Immunohistochemical staining with the five proteins showed no single protein to be a predictor of response to chemotherapy. However, pre-treatment breast cancer specimens that were annexin-A2 positive but annexin-A1 negative correlated with a poor pathological response (p = 0.04, Fisher’s exact test). The mechanisms by which annexins confer chemoresistance have not been identified, but may be due to inhibition of apoptosis. Annexin-A1 has been shown to enhance apoptosis, whilst annexin-A2, by contrast, inhibits apoptosis.

Introduction

Neoadjuvant chemotherapy (NAC) is being used increasingly in women with breast cancer, particularly in those who have large [(L) 3–5 cm] or locally advanced [(LA) T3, T4, N2] breast cancers (BCs).1 The advantages of NAC are to downstage the BCs, thereby, increasing the likelihood of breast conserving surgery (up to 50%) and reducing systemic micrometastatic tumour load, and thus possibly improving long-term survival.2, 3 Moreover, NAC can provide an opportunity to assess the likely outcome in any subsequent adjuvant therapeutic setting.4 NAC treatment with 4 cycles of adriamycin and cyclophosphamide, followed subsequently by 4 cycles of docetaxel, has demonstrated a good overall clinical response (up to 90%).4 However, the complete pathological response (cPR) rate is far less (up to 30%).4, 5, 6 NAC is a toxic and expensive treatment. Therefore, it would be clinically very beneficial to patients and satisfying to medical personnel if it was possible to predict the likelihood of response to NAC, thereby, selectively targeting treatment to those likely to benefit and preventing those patients unlikely to respond from undergoing expensive, toxic, prolonged and unnecessary treatment. However, to date, there is no study that has reported accurate and reliable markers to predict pathological response to NAC.

Previously, we had documented in vitro a group of proteins, using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry, demonstrating differential expression in chemosensitive and chemoresistant MCF-7 breast cancer cell lines.7 These proteins suggested a possible prediction profile in women with breast cancer, who were about to undergo NAC. In the study reported here, five proteins of interest, which are selected based on the results obtained from our previous proteomic studies and hypothesised to be putative biomarkers of prediction of response to chemotherapy, were investigated using immunohistochemical (IHC) staining of pre-treatment core needle biopsies (CNBs) from human breast cancers. These included proteins showing up- or down-regulation, proteins common to the two breast cancer cell lines (resistant to adriamycin and paclitaxel), proteins to which antibodies were commercially available for the research use in human tissues, proteins associated with different functions, as well as proteins that had not previously been documented in published studies. The results of the IHC staining in the pre-treatment tissues were correlated with the pathological response rates obtained after completion of the NAC.

Section snippets

Cell lines and cell clot

The MCF-7 wild type (MCF-7/WT, human breast adenocarcinoma) cell line was purchased from the European Collection of Cell Cultures (ATCC Number HTB-22). The MCF-7 adriamycin-resistant subline (MCF-7/ADR) and the MCF-7 paclitaxel-resistant subline (MCF-7/PAC) were kind gifts from Dr. Timothy Gant (MRC Toxicology Unit, University of Leicester, UK)8 and Dr. Susan Bates (National Cancer Institute, USA),9 respectively. All cell lines were grown, subcultured and tested for resistance to

IHC staining of MCF-7 cell clots

The results of staining of the MCF-7 cell clots are shown in Table 3, and their representative images (20× microscopic examination) are shown in Fig. 1A and B. Annexin-A1, annexin-A2, sorcin and stathmin were found to be over-expressed in chemoresistant MCF-7 cells than in chemosensitive MCF-7 cells. In contrast, HSP27 was found to be under-expressed. These findings were comparable to our previous proteomic study.7

Clinical characteristics versus pathological response and expression of annexins

There was no correlation between the pathological response documented and the

Discussion

Five proteins, significantly over- or under-expressed in our previously reported in vitro study on breast cancer cell lines resistant to chemotherapy, were selected for IHC staining.7 There is an ongoing debate about the reliability of using cancer cell lines as a model for biomolecular cancer research. Breast cancer cell lines have been widely used to investigate various biological mechanisms and signalling pathways, including proliferation, apoptosis, invasion and metastasis of breast cancer

Conflict of interest statement

None declared.

Acknowledgement

This study is funded by the Nottinghamshire-Derbyshire-Lincolnshire Research Alliance, UK.

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