Cancer Therapy: Clinical Association of Breast Cancer Stem Cells Identified by Aldehyde Dehydrogenase 1 Expression with Resistance to Sequential Paclitaxel and Epirubicin-Based Chemotherapy for Breast Cancers

Purpose: Breast cancer stem cells have been shown to be associated with resistance to chemotherapy in vitro, but their clinical significance remains to be clarified. The aim of this study was to investigate whether cancer stem cells were clinically significant for resistance to chemotherapy in human breast cancers. Experimental Design: Primary breast cancer patients (n = 108) treated with neoadjuvant chemotherapy consisting of sequential paclitaxel and epirubicin-based chemotherapy were included in the study. Breast cancer stem cells were identified by immunohistochemical staining of CD44/CD24 and aldehyde dehydrogenase 1 (ALDH1) in tumor tissues obtained before and after neoadjuvant chemotherapy. CD44/CD24 tumor cells or ALDH1-positive tumor cells were considered stem cells. Results: Thirty (27.8%) patients achieved pathologic complete response (pCR). ALDH1positive tumors were significantly associated with a low pCR rate (9.5% versus 32.2%; P = 0.037), but there was no significant association between CD44/CD24 tumor cell proportions and pCR rates. Changes in the proportion of CD44/CD24 or ALDH1positive tumor cells before and after neoadjuvant chemotherapy were studied in 78 patients who did not achieve pCR. The proportion of ALDH1-positive tumor cells increased significantly (P < 0.001) after neoadjuvant chemotherapy, but that of CD44/CD24 tumor cells did not. Conclusions:Our findings suggest that breast cancer stemcells identified asALDH1positive, but not CD44/CD24, play a significant role in resistance to chemotherapy. ALDH1positive thus seems to be a more significantly predictive marker than CD44/CD24 for the identification of breast cancer stem cells in terms of resistance to chemotherapy. Cancer stem cells are defined as rare tumor cells that are capable of self-renewal and give rise to multipotent progenitor cells, which ultimately differentiate into all cell types within the tumor (1–4). The cancer stem cell population is believed to be small, accounting for only 0.1% to 1% of all tumor cells. Cancer stem cells were first documented in acute myeloid leukemia by taking advantage of cell sorting technology using various surface markers (5). Later studies of solid tumors, including breast tumors, brain tumors, lung tumors, and colon tumors, have indicated the presence of cancer stem cells in these tumors as well (6–9). With respect to breast cancer, Al-Hajj et al. were the first to distinguish tumorigenic cancer cells (stem cells) from nontumorigenic ones by using cell surface markers CD44 and CD24 (6). They showed that as few as 100 tumor cells with CD44/CD24 phenotype were able to produce tumors in immunodeficient mice, whereas tumor cells with other CD44/CD24 phenotypes were unable or rarely able to produce tumors even when as many as 10 to 10 tumor cells were inoculated into such mice. Furthermore, Abraham et al. conducted immunohistochemical studies of CD44/CD24 tumor cells in human breast tumors and showed that breast tumors containing a high proportion of CD44/CD24 cells were associated with distant metastases (10). Recently, Ginestier et al. showed that aldehyde dehydrogenase 1 (ALDH1) is a better marker of breast cancer stem cells based on the finding that fewer ALDH1-positive than Authors' Affiliation: Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Received 6/12/08; revised 2/5/09; accepted 3/15/09; published OnlineFirst 6/9/09. Grant support: Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan and Promotion of Cancer Research (Japan) for the Third-Term Comprehensive 10-Year Strategy for Cancer Control. The costs of publication of this articlewere defrayed in part by thepayment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Shinzaburo Noguchi, Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan. Phone: 81-6-6879-3772; Fax: 81-6-6879-3779; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-1479 4234 Clin Cancer Res 2009;15(12) June 15, 2009 www.aacrjournals.org Research. on April 19, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from CD44/CD24 tumor cells are required to produce tumors in immunodeficient mice (11). In addition, they have been able to show that immunohistochemically identified ALDH1 expression is associated with poor prognosis in human breast cancers. ALDH1 in cancer stem cells may be a significant enzyme in stem cell differentiation that regulates the conversion of retinoic acid to oxidizing retinol (12). The results of Abraham et al. (10) and Ginestier et al. (11) seem to point to the existence of breast cancer stem cells and their association with a biologically aggressive phenotype. Another important characteristic of cancer stem cells is that they usually express high levels of ATP-binding cassette transporters and thus are thought to be resistant to various chemotherapeutic agents effluxed by ATP-binding cassette transporters (13, 14). In fact, several in vitro studies have shown that cancer stem cells are resistant to paclitaxel, doxorubicin, 5fluorouracil, and platinum (15–18). The implication that breast tumors may contain stem cells, which are supposedly resistant to chemotherapy, can be of major clinical importance for a better understanding of the mechanism of acquisition of drug resistance. Almost all breast tumors, although initially may respond to a given chemotherapy, ultimately become resistant to the chemotherapy. It is generally thought that tumor regrowth during chemotherapy results from clonal selection of tumor cells, which acquire their resistant properties due to various genetic/epigenetic mechanisms during the treatment (2). In the case of stem cells, however, it is considered that chemotherapy-resistant stem cells have been already present before chemotherapy and that tumor regrowth is attributable to the preferential proliferation of these stem cells. Taking all these findings into account leads to the speculation that breast tumors with a high proportion of stem cells may be associated with resistance to chemotherapy and that the proportion of stem cells may increase after chemotherapy because they are resistant to chemotherapy. In the study presented here, we investigated the validity of these speculations in a neoadjuvant chemotherapy setting in human breast cancers. We employed the two methods for the identification of breast cancer stem cells, CD44/CD24 and ALDH1, to compare their clinical utility for the prediction of resistance to chemotherapy. Materials and Methods Patients and breast tumor tissues. The subjects recruited for this study comprised 108 primary invasive breast cancer patients (mean age, 50.8 years; range, 26-72 years) with a tumor >3 cm in diameter or with cytologically confirmed axillary lymph node involvement who were treated with neoadjuvant chemotherapy at Osaka University Hospital between June 2003 and April 2007 (4 stage IV patients with small distant metastases were included in these subjects). Tumor specimens were obtained before neoadjuvant chemotherapy by means of vacuumassisted core needle biopsy. All patients were treated with 12 cycles of paclitaxel (80 mg/m/wk) followed by 4 cycles of 5-fluorouracil 500 mg/m, epirubicin 75 mg/m, and cyclophosphamide 500 mg/m every 3 weeks. Breast conserving surgery or mastectomy was conducted 3 to 4 weeks after the last treatment. Tumor specimens (surgical specimens) were also obtained at surgery. Informed consent was obtained