CD44+ cells in breast cancer are associated with worse prognosis and poor clinical outcome

Experimental and Molecular Pathology 100 (2016) 145–150

Contents lists available at ScienceDirect

Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp

ALDH+/CD44+ cells in breast cancer are associated with worse prognosis and poor clinical outcome Yan Qiu a,b, Tianjie Pu a,b, Peng Guo a, Bing Wei b, Zhang Zhang b, Hongying Zhang b, Xiaorong Zhong c, Hong Zheng c, Lina Chen d, Hong Bu a,b, Feng Ye a,⁎ a

Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China Cancer Center and Laboratory of Molecular Diagnosis of Cancer, State Key Laboratory of Biotherapy, National Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China d Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China b c

a r t i c l e

i n f o

Article history: Received 14 August 2015 Accepted 30 November 2015 Available online 11 December 2015 Keywords: Breast neoplasm Breast cancer stem cell ALDH+/CD44+ cells Metastasis Poor prognosis

a b s t r a c t Background: Breast cancer stem cells (BCSCs) play essential roles in tumor metastasis and contribute to remarkably negative clinical outcomes. Recently, aldehyde dehydrogenase (ALDH) and CD44 positivity (ALDH+/CD44+) was identified as a marker of BCSCs in vitro/in vivo studies. The aim of this study was to evaluate the prevalence of ALDH+/CD44+ cells in breast cancer and the association of these two markers with clinicopathological features and clinical outcomes. Materials and methods: We investigated the prevalence of ALDH1A3+/CD44+ cells in a cohort of 144 formalinfixed, paraffin-embedded (FFPE) breast cancer tissues. The tissues were stained for ALDH1A3 and CD44 by single and dual immunohistochemistry (dIHC). The associations among the prevalence of ALDH1A3+/CD44+ cells, the clinicopathological features and the clinical outcomes of the patients were also analyzed. Results: ALDH1A3+/CD44+ cells were present in 39 patients (27.1%). By the Mann–Whitney U test, the Pearson Chi-square test or Fisher's exact test, it was demonstrated that the prevalence of ALDH1A3+/CD44+ cells was closely correlated with larger tumor size (p = 0.001), nodal metastasis status (p = 0.043), more advanced clinical stage (p = 0.021) and distant metastasis after initial surgery (p = 0.001). In a univariate survival analysis, the presence of ALDH1A3+/CD44+ tumor cells had a significant negative association with both disease-free survival (DFS) and overall survival (OS) (pDFS b 0.001; pOS b 0.001). The negative clinical outcomes in ALDH1A3+/CD44+ tumors were further confirmed by a multivariate analysis using Cox proportional hazard models (pDFS b 0.001, HR = 3.155; pOS = 0.001, HR = 3.193). This was also true with respect to the clinical treatment regimens of chemotherapy (pDFS b 0.001; pOS = 0.001), radiotherapy (pDFS = 0.004; pOS = 0.004), and endocrine therapy (pDFS b 0.001; pOS b 0.001). Conclusion: In summary, our results indicate that the prevalence of ALDH1A3+/CD44+ tumor cells in breast cancer is significantly associated with worse prognostic factors and favors a poor prognosis. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Breast cancer is currently considered to include heterogeneous tumors with diverse biological behaviors, clinical outcomes, and responses to treatment (Jemal et al., 2011). Despite various therapies that have been applied to treat breast malignancies, breast cancer is still the leading cause of death for women worldwide (Jemal et al., 2010). In recent years, increasing evidence has suggested that only a small subset of cells, which are defined as breast cancer stem cells (BCSCs), is responsible for the heterogeneity (Ginestier et al., 2007), aggressive behavior and therapeutic resistance in breast cancer (Lee et al., ⁎ Corresponding author. E-mail address: [email protected] (F. Ye).

http://dx.doi.org/10.1016/j.yexmp.2015.11.032 0014-4800/© 2015 Elsevier Inc. All rights reserved.

2011; Velasco-Velázquez et al., 2012). Therefore, CSCs are potential prognostic indicators in patients with breast cancer (Dalerba et al., 2007; Tsang et al., 2012). During the past decade, much effort has been expended to define biomarkers that may enable the identification of CSCs. Various proteins such as CD44, CD24, ALDH1, CD133, CD49 and EpCAM have been identified as biomarkers of breast CSCs, which have been found to demonstrate CSC characteristics in breast neoplasm (Visvader and Lindeman, 2008; Vieira et al., 2012). To identify CSCs with greater specificity, the use of the co-expression of markers such as CD44/CD24 (Al-Hajj et al., 2003; Abraham et al., 2005), CD49/EpCAM (Ghebeh et al., 2013), and CD44/ALDH (Croker et al., 2009) has been advocated. However, the ideal combination of markers has not yet to be confirmed for the identification of CSCs that are capable of the initiation and metastasis of

146

Y. Qiu et al. / Experimental and Molecular Pathology 100 (2016) 145–150

tumors. Croker and his colleagues demonstrated that high ALDH expression and CD44 expression in cells of mice and in vitro experiments enhanced the invasion and metastatic ability of the tumor cells (Croker et al., 2009) and that the inhibition of ALDH activity could reduce the resistance of ALDHhiCD44+ cells to chemotherapy and radiotherapy (Croker and Allan, 2012). More recently, Liu et al. (2013) demonstrated that ALDHhigh/CD44high cell subsets showed the highest enhancement of stem cell phenotypic properties compared to ALDHhigh/CD44low, ALDHlow /CD44high, ALDHlow/CD44low and unsorted controls. In a recent study, Cui et al. (2015)revealed that the mRNA levels of Notch1 and β-catenin were significantly higher in ALDHhiCD44+ cells compared with those in ALDHlowCD44+ cells and found a high correlation between ALDHhi CD44+ cells and Ki-67 expression; however, they did not find an association between ALDHhiCD44+ cells and ER, PR and HER2 expression. Moreover, they suggested that the presence of ALDHhiCD44+ cells might serve as a novel diagnostic and prognostic factor in breast cancer. In clinical breast cancers, CD44 and ALDH1 have both been reported as predictors of poor clinical outcomes (Ginestier et al., 2007; Rovira et al., 2010; McFarlane et al., 2015.), but the prevalence of ALDH+ CD44+ cells and the relationship between the level of expression of these two markers and clinical outcomes have never been clarified. In our previous study, we showed that out of all the proteins in the ALDH family, ALDH1A3 might be responsible for the poor outcomes of patients with breast cancer (Qiu et al., 2014). In this study, we enrolled 144 patients with invasive ductal breast cancer (IDC) and evaluated the prevalence of ALDH1A3 and CD44 co-expression in tumor cells (ALDH1A3+ CD44+) and the association of these markers with clinicopathological parameters, distant metastasis, recurrence and survival. Our data demonstrated that patients with ALDH+/CD44+ tumor cells were more likely to have distant metastases. Moreover, we showed that disease-free survival and overall survival were decreased in patients with ALDH+/CD44+ cells, which suggests that the presence of ALDH+ CD44+ cells is an independent predictive indicator of poor outcomes in IDC. 2. Materials and methods 2.1. Patients and sample preparation Specimens from 144 patients with IDC were surgically removed between 2006 and 2009. All specimens were collected and diagnosed in the Department of Pathology of West China Hospital. We prepared tissue microarray (TMA) cores of 1.5 mm from the FPPE samples. Two cores from each individual tumor were arrayed. All patients were female (median age, 51 years; range, 33–77 years), were diagnosed on the basis of histological findings and were staged according to the TNM system. The follow-up time ranged from 2 to 102 months (median, 84.3 months). DFS and OS were defined as the time between the initial surgery and local or distant metastatic relapse, and the time between surgery and death, respectively. Approval for the study was granted by the Ethics Committee of West China Hospital (No. 2013-191). 2.2. Immunohistochemistry (IHC) Dual IHC with antibodies for the detection of CD44 and ALDH1A3 was performed with the EnVision G | 2 Doublestain System Rabbit/ Mouse (DAB +/Permanent Red) according to the manufacturer's instructions. Sections with a thickness of 0.4 μm were cut from paraffin-embedded TMA blocks and were mounted on adhesivecoated glass slides. The TMA slides were deparaffinized and rehydrated in dimethylbenzene and ethanol, respectively. Endogenous peroxidase was blocked by 3% H2O2, and epitope retrieval was performed in a pressure sterilizer. After blocking with 10% serum for 20 min at room temperature (RT), the slides were further incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD44 (1:200; 04-1123, Merck Millipore, Darmstadt, Germany) and mouse anti-ALDH1A3

(1:400; Origene, TA502805, Rockville, USA). After 5 washes in phosphate-buffered saline, the slides were incubated with anti-rabbit and anti-mouse secondary antibodies for 30 min at room temperature. CD44 was detected with Permanent Red while ALDH1A3 was detected with diaminobenzidine (DAB). Single IHC with anti-CD44 and antiALDH1A3 antibodies was also performed as a control. The hematoxylin and eosin (H&E) as well as the IHC stains were assessed by light microscopy. The staff pathologist at West China Hospital conducted a standard pathological assessment of the tumors from the anonymous patient panel. The status of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in the specimens was obtained from the initial pathology reports. HER2 staining was analyzed according to the guidelines of the American Society of Clinical Oncology. CD44 staining was detected primarily in the membrane, and the scoring was performed as follows: 0, 0% positive tumor cells; 1, 1% to 10% positive cells; 2, 11% to 50% positive cells; 3, 51% to 75% positive cells; and 4, 76% to 100% positive cells. ALDH1A3 staining was detected primarily in the cytoplasm, and the scoring was performed as described for CD44 (Honeth et al., 2008). 2.3. Statistical analysis The associations between the presence of ALDH1A3+/CD44+, the clinical variables and the breast cancer subgroups were assessed by Pearson's Chi-square test and Fisher's exact test. Survival curves were plotted according to the Kaplan–Meier method and were compared by log-rank test. The significance of the various parameters for survival was analyzed by the Cox proportional hazards model in a multivariate analysis. Statistical analyses were performed with SPSS version 16.0 software (SPSS Inc. Chicago, USA). 3. Result 3.1. Co-expression of ALDHA3 and CD44 in invasive ductal breast cancer In order to clearly identify the target tumorigenic cells, we performed both single IHC and dual IHC (Fig. 1A–D) on our TMA samples. In the breast tumor cells, CD44 is predominantly expressed on the membrane, whereas ALDH1A3 is mainly present in the cytoplasm. ALDH1A3+/CD44+ cells were present in 39 out of 144 (27.1%, Table 1) patients while ALDH1A3+/CD44+ tumor cells ranged between 0% and 80%; the results of the single IHC assay demonstrated that ALDH1A3+ and CD44+ cells ranged from 0 to 100% (data not shown). 3.2. Baseline clinical characteristics of the tumor tissues The impact of the clinicopathologic characteristics and prognostic factors was calculated by Kaplan–Meier analysis, and the survival curves were delineated by log-rank test. We observed that larger tumor size (pDFS = 0.014, pOS = 0.003), increased nodal status (pDFS b 0.001, pOS b 0.001), clinical stage (pDFS = 0.013, pOS b 0.001), and the presence of distant metastasis (pDFS b 0.001, pOS b 0.001) or recurrence (pDFS b 0.001, pOS = 0.015) all had a close association with poor clinical outcomes. Twelve patients (8.3%) had recurrences, and detailed information in regard to tumor recurrence was available for all patients. Additionally, 59 (40.1%) patients had distant metastases, which were primarily observed in the bone, liver, lungs, and other organs (Table 2). 3.3. Correlation of the prevalence of ALDH1A3+/CD44+ cells with tumor characteristics We compared the expression status of ALDH1A3+/CD44+ in different subgroups of invasive breast cancer, which were stratified by clinical characteristics and prognostic factors (i.e., WHO grade, tumor size, nodal status, ER status, PR status, HER2 status, clinical stage, distant metastasis and relapse) by Mann–Whitney U and Pearson Chi-square tests

Y. Qiu et al. / Experimental and Molecular Pathology 100 (2016) 145–150

147

Fig. 1. Dual-IHC and immunofluorescence staining on TMA slides. Dual-IHC staining with primary antibodies of ALDHA3 and CD44 was performed on TMA tissue. A, patients whose negative for both ALDH and CD44; B, patient shows only CD44 positive cancer cells (membrane); C, patient shows only ALDH1A3 positive cancer cells (cytoplasm); D, case with cancer cell that expresses both biomarkers.

or by Fisher's exact test. No statistically significant differences were observed between patients with the presence of ALDH1A3+/CD44+ tumor cells and patients with the absence of ALDH1A3+/CD44+ tumor cells when these two groups were stratified by those factors except for tumor size, clinical stage and distant metastasis. The results showed that the prevalence of ALDH1A3+/CD44+ tumor cells was more likely to be observed in patients with distant metastasis (64.2%, p = 0.001), larger tumor size (p = 0.001), greater nodal status (p = 0.043), and more advanced clinical stage (p = 0.021) (Table 3). Furthermore, our discovery revealed no correlation between the prevalence of ALDH1A3+/ CD44+ cells and hormone receptor status(ER status, PR status and HER2 status), which suggests a lack of an association between molecular subtypes and the prevalence of ALDH1A3+/CD44+ cells. The staining of the samples for ALDH1A3+ (50.1%, p = 0.002) and CD44+(59.7%, p = 0.001) cells also revealed a significant correlation with distant metastasis. However, these correlations were no more significant than when the tumor samples were stained for ALDH1A3+/CD44+ cells (Supplemental Tables 2 and 3). 3.4. Association between the prevalence of CD44+, ALDH1A3+ and ALDH1A3+/CD44+ cells and survival To evaluate whether CD44 and ALDH1A3 co-expression correlated with progression (recurrence and distant metastasis) and death in

Table 1 The prevalence of ALDH1A3+, CD44+, and ALDH1A3+/CD44+ cells in IDC. IHC

Score

CD44+

ALDH1A3+

ALDH1A3+/CD44+

Negative Positive

0 1 2 3 4

79 (54.9) 27 (18.8) 24 (16.7) 11 (7.6) 3 (2.1)

54 (37.5) 1 (3.5) 5 (5.6) 7 (4.9) 77 (53.5)

105 (72.9) 17 (11.8) 15 (10.4) 6 (4.2) 1 (0.7)

patients with breast cancer, univariate and multivariate survival analyses were applied. We performed a survival analysis to identify the correlation between survival and the prevalence of ALDH1A3+/CD44+ Table 2 Baseline clinical characteristics of the study subjects.

N% WHO grade 1 2 3 Tumor size ≤5 cm N5 cm Nodal status N0–1 N2–3 Clinical stage I–II III–IV ER status Negative Positive PR status Negative Positive HER2 status 0/1+ 2+ 3+ Distant metastasis No Yes Recurrence No Yes

144 5 (3.5) 39 (27.1) 100 (69.4) 144 113 (78.4) 31 (21.6) 144 86 (59.7) 58 (40.3) 144 78 (53.1) 66 (45.8) 144 53 (36.8) 91 (63.2) 144 54 (37.5) 90 (62.5) 144 126 (87.5) 12 (8.3) 6 (4.2) 144 85 (59.0) 59 (41.0) 132 (91.7) 12 (8.3)

⁎ p value is statistically significant.

Disease-free survival

Overall survival

Log-rank

p-Value

Log-rank

p-Value

3.594

0.166

5.651

0.059

6.007

0.014⁎

8.924

0.003⁎

13.100

b0.001⁎

19.227

b0.001⁎

6.227

0.013⁎

12.448

b0.001⁎

0.119

0.731

2.901

0.089

5.657

0.017⁎

6.702

0.010⁎

3.690

0.518

1.894

0.388

151.214

b0.001⁎

72.287

b0.001⁎

43.000

b0.001⁎

5.914

0.015⁎

148

Y. Qiu et al. / Experimental and Molecular Pathology 100 (2016) 145–150

Table 3 Correlation of the prevalence of ALDH1A3+/CD44+ cells and tumor characteristics. ALDH1A3+/CD44+ cells

WHO grade 1 2 3 Tumor size ≤5 cm N5 cm Nodal status N0–1 N2–3 Clinical stage I–II III–IV ER status Negative Positive PR status Negative Positive HER2 status 0/1+ 2+ 3+ Distant metastasis No Yes Recurrence No Yes

Negative

Positive

p-Value

105 3 (2.9) 27 (25.7) 75 (71.4) 105 90 (85.7) 15 (4.3) 105 68 (64.8) 37 (35.2) 105 63 (60.0) 42 (40.0) 105 41 (39.0) 64 (61.0) 105 36 (34.3) 69 (65.7) 105 93 (88.6) 7 (6.7) 5 (4.7) 105 71 (67.6) 34 (32.3) 105 95 (90.5) 10 (95.2)

39 2 (5.1) 12 (30.8) 25 (64.1) 39 23 (60.0) 16 (40.0) 39 18 (46.2) 21 (53.8) 39 15 (38.5) 24 (61.5) 39 12 (30.8) 27 (69.2) 39 18 (46.2) 21 (53.8) 39 33 (84.6) 5 (12.8) 1 (2.6) 39 14 (35.9) 25 (64.1) 39 37 (94.8) 2 (5.2)

0.634

0.001⁎

0.043⁎

0.021⁎

0.360

0.191

0.433

0.001⁎

0.396

⁎ p value is statistically significant.

cells. Significantly reduced DFS and OS were observed in breast cancer patients with a prevalence of ALDH1A3+/CD44+ (pDFS b 0.001, pOS b 0.001) tumor cells (Fig. 2 and Supplemental Table 3). In a multivariate analysis using the Cox method, WHO grade, tumor size, nodal status, primary metastasis status, ER, PR, and Her2 status were enrolled as covariates of ALDH1A3+/CD44+ status. We found that the prevalence of ALDH1A3+/CD44+ (pDFS b 0.001, hazard risk = 3.155; pOS = 0.001, HR = 3.193) cells was an independent and protective prognostic marker for the progression (recurrence and distant metastasis) of breast cancer. Furthermore, we analyzed the association between the presence of CD44+ cells and survival as well as the presence of ALDH1A3+ cells and survival. A univariate analysis showed that the presence of ALDH1A3+ (pDFS = 0.001, pOS = 0.005, Supplemental Fig. 1) and CD44+ (pDFS b 0.001, pOS b 0.001, Supplemental Fig. 2) cells in patients was associated with decreased DFS and OS. In addition, a multivariate analysis suggested that ALDH1A3+ (pDFS = 0.015, hazard ratio (HR) = 2.002; pOS = 0.004, HR = 2.662) and CD44+(pDFS = 0.010, HR = 2.363; pOS = 0.23, HR = 2.498) cells contribute to poor outcomes in patients

with IDC. Obviously, the relative risk (hazard risk) for the occurrence of progression and death was the highest in patients with ALDH1A3+/ CD44+ tumor cells (Table 4). With respect to different therapies, the presence of ALDH1A3+/ CD44+ tumor cells still demonstrated a close association with metastasis, disease-free survival (Table 5), and overall survival. This was also true for chemotherapy, radiotherapy and hormonal treatment. 4. Discussion As has been widely accepted, metastasis and not the initial tumor, is the primary cause of death of patients with breast malignancies. An increasing number of studies have shown that an enlarged cancer stem cell population is highly associated with tumor metastatic ability and that the cancer stem cells can be expanded to drive cancer progression and motility. Although there is a degree of consensus on the use of certain proteins as BCSC markers, some controversy persists, and therefore, the need to identify further markers is warranted. In pioneering studies by Al-Hajj et al., CD44+/CD24−/low cells were identified as prospective cancer stem cells in breast cancer. Subsequently, ALDH, which comprises a family of evolutionarily conserved enzymes, was shown to possess stem cell-like abilities by both in vitro and in vivo experiments (Ginestier et al.,2007). Since then, a large number of biological and clinical studies of CD44 and ALDH1 have appeared to verify the conclusions of earlier studies. However, their conclusions were inconsistent. Although Lopez et al. (2005) demonstrated that CD44 can inhibit metastasis in breast cancer, most studies have demonstrated its pivotal role in the promotion of metastasis (Bourguignon, 2001; Montgomery et al., 2012). Similar to CD44, the activity of ALDH1 is also associated with controversial study results (Marcato et al., 2011a, b). ALDH1A3, one of nineteen isoforms of the ALDH family, was found to dominantly contribute to ALDEFLUOR activity, and its specific expression was suggested to be predictive of tumor grade, cancer stage and metastasis in patients with breast cancer (Qiu et al., 2014; Marcato et al., 2011a, Eirew et al., 2012; Marcato et al., 2011b) In the present study, we combined these two markers, and the results showed that the prevalence of ALDH1A3+/CD44+ cells was associated with poor prognostic parameters such as increased tumor size, higher nodal status and clinical stage. Critically, it closely correlated with the occurrence of distant metastasis. As expected, compared with patients whose tumors expressed a single marker, distant metastasis rate (ALDH1A3 (59.7%) or CD44 (50.1%)) was more likely to be found in patients whose tumors coexpressed these two markers (64.2%) (Table 2 and Supplemental Tables 2, 3). This indicates that ALDH1A3+/CD44+ might be a much more sensitive marker for the prediction of metastasis than either ALDH or CD44 alone. Our findings are in disagreement with the results of a previous study by Paola Marcato and his group (Carrasco et al., 2014). They indicated that the prognostic potential of the combination of ALDH1A3 and CD44 expression in cells had shown inferiority to ALDH1A3 expression alone. From our viewpoint, our data were much

Fig. 2. Correlation of the prevalence of ALDH1A3+/CD44+ cells with breast cancer prognosis (univariate analysis).

Y. Qiu et al. / Experimental and Molecular Pathology 100 (2016) 145–150

149

Table 4 Correlation of the prevalence of ALDH1A3+, CD44+, ALDH1A3+/CD44+ cells with breast cancer prognosis (multivariate analysis).

N (%) ALDH1A3 −(0%) +(N0%) CD44 −(0%) +(N0%) ALDH1A3+/CD44+ −(0%) +(N0%)

Disease-free survival

Overall survival

p value

HR (95% CI)

p value

0.010⁎

144 53 91 144 79 65 144 105 39

HR (95% CI)

0.023⁎ 1 2.363 (1.231–4.536)

0.015⁎

1 2.002 (1.144–3. 503)

b0.001⁎

1 2.498 (1.133–5.508)

0.004⁎

1 2.662 (1.362–5.201)

0.001⁎

1 3.155 (1.727–5.763)

1 3.193 (1.576–6.509)

⁎ p value is statistically significant.

more persuasive because our study included approximately 3-fold more patients than theirs. Furthermore, according to a survival analysis, the results showed that patients whose tumors co-expressed ALDH1A3 and CD44 were prone to have shorter disease-free survival and overall survival (Table 3 and Fig. 2) than those whose tumors did not co-express these markers. In addition, according to a multivariate analysis, the relative risk for the occurrence of progression and death in patients with ALDH1A3+/CD44+ cells was 3.16- and 3.19-fold greater, respectively, than those without ALDH1A3+/CD44+ cells. Our clinical data indicated that ALDH1A3+/CD44+ might be used as a biomarker to predict patients who are at an increased risk for progression (metastasis or relapse) and death. In a previous study, ALDHhi/CD44+ was identified as a marker of CSCs in breast cancer; this study also showed that ALDHhi/ CD44+ cells could promote malignant and metastatic behavior in breast cancer (Croker et al., 2009).Coincidently, similar findings were reported in lung cancer by Liu and his colleagues, who demonstrated that lung cancer tumorigenicity is enhanced through ALDHhi/CD44hi tumorinitiating cells (Liu et al., 2013). Our data confirmed previous findings and linked the development of metastatic breast cancer with the appearance of CSCs. Our data and previous findings showed that ALDH1A3+/CD44+ cells are likely CSCs in breast cancer. As hypothesized, the CSCs that are ALDH1A3+/CD44+ may be the chief culprits of metastasis and therapeutic resistance in breast cancer and other solid tumors. Overcoming therapeutic resistance is crucial if breast cancer is to be cured. Various studies have demonstrated that breast CSCs contribute to radioresistance and chemoresistance, (Carrasco et al., 2014) and many targeted therapies have thus been designed to differentiate or eradicate CSCs to reduce cancer metastases and elongate the overall survival of patients. Gupta et al. discovered a special compound named salinomycin, which can induce the epithelial differentiation of tumor cells (Gupta et al., 2009). In another study, Ginestier and his group suggested a novel means to target and eliminate breast CSCs via blockage of the IL8 receptor CXLR1 (Ginestier et al., 2010). In addition, published studies have shown that suppressed Stat-3 signaling pathway through inhibiting STAT3 and

other Stat-3 signaling mediators might also be potential breast CSCs targets (Su et al., 2012; Gopalan et al., 2013; Thakur et al., 2015). ALDH1A3+/CD44+, as a possible CSC marker, might be used as a novel target for a more effective treatment of breast cancer. We may be able to use specific methods to differentiate the ALDH1A3+/CD44+ cells into mature cancer cells so that their sensitivity to traditional therapies may be enhanced. Alternatively, we may be able to suppress ALDH activity and knock down the CD44 gene to inhibit the aggressive behavior of these tumors or induce the tumor cells to undergo apoptosis. It has been reported that diethylaminobenzaldehyde (DEAB) could directly reduce ALDH activity, but data published thus far have not yet confirmed the benefit of these approaches in terms of breast cancer prognosis. This is still a controversial issue and therefore further studies are required. In conclusion, our data revealed that the prevalence of ALDH1A3+/ CD44+ cells is an independent predictive indicator of metastasis and poor prognosis in patients with invasive ductal breast cancer. In addition, when combined with data from previous studies, the prevalence of ALDH1A3+/CD44+ cells, as a possible marker of breast CSCs, may provide a novel potential target for a therapeutic approach that may eliminate breast tumors and minimize the potential for metastasis or recurrence. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yexmp.2015.11.032. Conflict of interest statement There are not any ethical/legal conflicts involved in the article. Acknowledgments This work was funded by grants from the National Natural Science Foundation of China (31000601), (81200461) and Young Investigator Scholar in Sichuan University (2012SCU04A14). Approval for the study was granted by the Ethics Committee of West China Hospital (No. 2013-191).

Table 5 Prevalence of ALDH1A3+/CD44+ tumor cells and treatment response. Recurrence Therapy

N

Chemotherapy 0% N0% Radiotherapy 0% N0% Endocrine therapy 0% N0%

131 95 36 86 79 7 69 61 8

⁎ p value is statistically significant.

No

Distant metastasis Yes

p value

No

Yes

0.379 85 (71.4) 10 (83.3)

34 (28.6) 2 (16.7)

57 (72.2) 5 (71.4)

22 (27.8) 2 (28.6)

48 (78.7) 7 (87.5)

13 (21.3) 1 (12.5)

61 (81.3) 34 (60.7)

14 (28.7) 22 (39.3)

45 (80.4) 17 (56.7)

11 (19.6) 13 (43.3)

39 (90.7) 16 (61.5)

4 (9.3) 10 (38.5)

0.967

0.560

Disease-free survival

Overall survival

p value

Log-rank

p value

Log-rank

p value

0.009

13.773

b0.001⁎

11.473

0.001

0.020

8.149

0.004

8.100

0.004⁎

0.005⁎

17.381

b0.001⁎

11.624

0.001⁎

150

Y. Qiu et al. / Experimental and Molecular Pathology 100 (2016) 145–150

References Abraham, B.K., Fritz, P., McClellan, M., Hauptvogel, P., Athelogou, M., Brauch, H., 2005. Prevalence of CD44+/CD24−/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin. Cancer Res. 11, 1154–1159. Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J., Clarke, M.F., 2003. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U. S. A. 100, 3983–3988. Bourguignon, L.Y., 2001. CD44-mediated oncogenic signaling and cytoskeleton activation during mammary tumor progression. J. Mammary Gland Biol. Neoplasia 6, 287–297. Carrasco, E., Alvarez, P.J., Prados, J., Melguizo, C., Rama, A.R., Aránega, A., RodríguezSerrano, F., 2014. Cancer stem cells and their implication in breast cancer. Eur. J. Clin. Investig. 44, 678–687. Croker, A.K., Allan, A.L., 2012. Inhibition of aldehyde dehydrogenase (ALDH) activity reduces chemotherapy and radiation resistance of stem-like ALDHhiCD44+ human breast cancer cells. Breast Cancer Res. Treat. 133, 75–87. Croker, A.K., Goodale, D., Chu, J., Postenka, C., Hedley, B.D., Hess, D.A., Allan, A.L., 2009. High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J. Cell. Mol. Med. 13, 2236–2252. Cui, J., Li, P., Liu, X., Hu, H., Wei, W., 2015. Abnormal expression of the Notch and Wnt/βcatenin signaling pathways in stem-like ALDH(hi)CD44(+) cells correlates highly with Ki-67 expression in breast cancer. Oncol. Lett. 9, 1600–1606. Dalerba, P., Cho, R.W., Clarke, M.F., 2007. Cancer stem cells: models and concepts. Annu. Rev. Med. 58, 267–284. Eirew, P., Kannan, N., Knapp, D.J., Vaillant, F., Emerman, J.T., Lindeman, G.J., Visvader, J.E., Eaves, C.J., 2012. Aldehyde dehydrogenase activity is a biomarker of primitive normal human mammary luminal cells. Stem Cells 30, 344–348. Ghebeh, H., Sleiman, G.M., Manogaran, P.S., Al-Mazrou, A., Barhoush, E., Al-Mohanna, F.H., Tulbah, A., Al-Faqeeh, K., Adra, C.N., 2013. Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers. BMC Cancer 13, 289. Ginestier, C., Hur, M.H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown, M., Jacquemier, J., Viens, P., Kleer, C.G., Liu, S., Schott, A., Hayes, D., Birnbaum, D., Wicha, M.S., Dontu, G., 2007. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567. Ginestier, C., Liu, S., Diebel, M.E., Korkaya, H., Luo, M., Brown, M., Wicinski, J., Cabaud, O., Charafe-Jauffret, E., Birnbaum, D., Guan, J.L., Dontu, G., Wicha, M.S., 2010. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J. Clin. Invest. 120, 485–497. Gopalan, A., Yu, W., Sanders, B.G., Kline, K., 2013. Eliminating drug resistant breast cancer stem-like cells with combination of simvastatin and gamma-tocotrienol. Cancer Lett. 328, 285–296. Gupta, P.B., Onder, T.T., Jiang, G., Tao, K., Kuperwasser, C., Weinberg, R.A., Lander, E.S., 2009. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138, 645–659. Honeth, G., Bendahl, P.O., Ringnér, M., Saal, L.H., Gruvberger-Saal, S.K., Lvgren, K., Grabau, D., Fern, M., Borg, A., Hegardt, C., 2008. The CD44+/CD24− phenotype is enriched in basal-like breast tumors. Breast Cancer Res. 10, R53. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. Global Cancer Statistics. CA Cancer J. Clin. 61, 69–90.

Jemal, A., Center, M.M., DeSantis, C., Ward, E.M., 2010. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol. Biomark. Prev. 19, 1893–1907. Lee, H.E., Kim, J.H., Kim, Y.J., Choi, S.Y., Kim, S.W., Kang, E., Chung, I.Y., Kim, I.A., Kim, E.J., Choi, Y., Ryu, H.S., Park, S.Y., 2011. An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer. Br. J. Cancer 104, 1730–1738. Liu, J., Xiao, Z., Wong, S.K., Tin, V.P., Ho, K.Y., Wang, J., Sham, M.H., Wong, M.P., 2013. Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells. Oncotarget 4, 1698–1711. Lopez, J.I., Camenisch, T.D., Stevens, M.V., Sands, B.J., McDonald, J., Schroeder, J.A., 2005. CD44 attenuates metastatic invasion during breast cancer progression. Cancer Res. 65, 6755–6763. Marcato, P., Dean, C.A., Giacomantonio, C.A., Lee, P.W., 2011a. Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle 10, 1378–1384. Marcato, P., Dean, C.A., Pan, D., Araslanova, R., Gillis, M., Joshi, M., Helyer, L., Pan, L., Leidal, A., Gujar, S., Giacomantonio, C.A., Lee, P.W., 2011b. Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis. Stem Cells 29, 32–45. McFarlane, S., Coulter, J.A., Tibbits, P., O'Grady, A., McFarlane, C., Montgomery, N., Hill, A., McCarthy, H.O., Young, L.S., Kay, E.W., Isacke, C.M., Waugh, D.J., 2015. CD44 increases the efficiency of distant metastasis of breast cancer. Oncotarget 6, 11465–11476. Montgomery, N., Hill, A., McFarlane, S., Neisen, J., O'Grady, A., Conlon, S., Jirstrom, K., Kay, E.W., Waugh, D.J., 2012. CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity. Breast Cancer Res. 14, R84. Qiu, Y., Pu, T., Li, L., Cheng, F., Lu, C., Sun, L., Teng, X., Ye, F., Bu, H., 2014. The expression of aldehyde dehydrogenase family in breast cancer. J. Breast Cancer 17, 54–60. Rovira, M., Scott, C.-J.E., Ginestier, C., Iovino, F., et al., 2010. Aldehyde dehydrogenase 1positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin. Cancer Res. 16, 45–55. Su, F., Ren, F., Rong, Y., Wang, Y., Geng, Y., Wang, Y., Feng, M., Ju, Y., Li, Y., Zhao, Z.J., Meng, K., Chang, Z., 2012. Protein tyrosine phosphatase Meg2 dephosphorylates signal transducer and activator of transcription 3 and suppresses tumor growth in breast cancer. Breast Cancer Res. 14, R38. Thakur, R., Trivedi, R., Rastogi, N., Singh, M., Mishra, D.P., 2015. Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer. Sci. Report. 5, 10194. Tsang, J.Y., Huang, Y.H., Luo, M.H., Ni, Y.B., Chan, S.K., Lui, P.C., Yu, A.M., Tan, P.H., Tse, G.M., 2012. Cancer stem cell markers are associated with adverse biomarker profiles and molecular subtypes of breast cancer. Breast Cancer Res. Treat. 136, 407–417. Velasco-Velázquez, M.A., Homsi, N., De La Fuente, M., Pestell, R.G., 2012. Breast cancer stem cells. Int. J. Biochem. Cell Biol. 44, 573–577. Vieira, A.F., Ricardo, S., Ablett, M.P., Dionísio, M.R., Mendes, N., Albergaria, A., Farnie, G., Gerhard, R., Cameselle-Teijeiro, J.F., Seruca, R., Schmitt, F., Clarke, R.B., Paredes, J., 2012. P-cadherin is coexpressed with CD44 and CD49f and mediates stem cell properties in basal-like breast cancer. Stem Cells 30, 854–864. Visvader, J.E., Lindeman, G.J., 2008. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat. Rev. Cancer 8, 755–768.