HER2-expressing breast tumors are associated with breast cancer stem-cell phenotype CD44+/CD24-

1. Pontifícia Universidade Católica do Paraná (PUCPR), Paraná, Brazil
2. Universidade Federal do Paraná (UFPR), Paraná, Brazil. 3. Universidade Positivo (UP), Paraná, Brazil

Endereço para correspondência

Iris Rabinovich
Av. Vereador Toaldo Túlio, 895, casa 9; Santa Felicidade
CEP: 82320-010; Curitiba-PR, Brasil
e-mail: iris_rbv@yahoo.com.br

INTRODUCTION

Breast cancer (BC) is the most commonly diagnosed malignancy in women^{(1, 2)} and the second most common cause of cancer death in females. It is recognized as a heterogeneous and phenotypically diverse disease characterized by great variability in the recurrence pattern and survival outcomes observed among different BC subtypes. The initial gene expression arrays based on the measurement of gene cluster identified distinct molecular subtypes such as luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and basal subtypes^(3-6). The luminal A subtype is enriched with genes similar to those found in luminal cells from the normal breast parenchyma and characterized by a high expression of hormone receptors and favorable outcome. The luminal B subgroup differs from luminal A in terms of high expression of proliferative genes, thereby presenting worse prognosis as compared with luminal A. HER2 and basal subtypes display poor prognosis in terms of overall survival and disease-free survival^(4-6). This analysis allowed the description of a tumor molecular portrait with different molecular subtypes harboring distinct prognosis^{(4, 5)} and showing different responses to local and systemic therapy^{(7, 8)}. Molecular classification is based on the differences in the pattern of gene expression in tumors. However, genetic array testing may not always be feasible in clinical practice. Hence, immunohistochemical analysis has been applied to molecular classification (Table 1)^(9-11). The 12^th St. Gallen International Breast Cancer Conference held in 2011⁽¹²⁾ introduced the molecular subtypes in BC treatment and allowed approximation of the molecular classification with clinicopathological criteria based on the immunohistochemistry analysis. It was recently demonstrated that the discordance level between molecular classification and clinicopathological criteria level may reach up to 30%⁽¹³⁾.

In a cancer stem-cell model, the tumor originates from a small population of stem cells that lose regulation of the selfrenewal process^{(14, 15)}. The consequences are the differentiation and growth of these cells accompanied by genetic and epigenetic alterations. Resistance to therapy may be associated with this clonal evolution⁽¹⁶⁾. Some of these cells may be identified by cell surface markers, such as the presence of CD44 and absence of CD24 markers (CD44⁺/CD24^– phenotype)⁽¹⁷⁾. CD44 and CD24 were shown to promote⁽¹⁸⁾ or inhibit⁽¹⁹⁾ invasion and metastasis of BC cells. However, the phenotype CD44⁺/CD24^– was clearly associated with invasion, as CD44⁺/CD24^– subpopulation was more invasive than CD44⁺/CD24^– subpopulation of the same TMD-436 cell line⁽²⁰⁾. CD44⁺/CD24^– phenotype was described as an indicator of stem cells in human breast tissue. These cells have been shown to survive cytotoxic therapy, leading to treatment failures and recurrences⁽²¹⁾, besides being closely correlated with the invasive property and poor prognosis⁽²²⁾. As cancer stem cells may be responsible for treatment resistance, these cell types are expected to be enriched in more aggressive subtypes, resulting in worse prognosis.

Here, we investigated the relationship between CD44⁺/CD24^– phenotype and molecular subtypes in BC using tissue microarray (TMA) technique. Furthermore, we examined the correlation of this phenotype with classical prognostic factors such as age, histological grade, tumor size, and lymph node status.

METHODS

Patients and specimens

Breast tumor samples of invasive ductal carcinoma from 133 patients who underwent surgery at the Breast Unit of Hospital Nossa Senhora das Graças (Curitiba, Brazil) between January 1998 and July 2009 were included in a non-consecutive manner. Patients that underwent mastectomy or breast-conserving surgery with sentinel node biopsy or axillary dissection and had a confirmed invasive ductal BC diagnosis were included in this study, while patients that underwent neoadjuvant chemotherapy or had insufficient paraffin-embedded tissue for the new analysis were excluded from the study.

Tissue microarray construction

Formalin-fixed paraffin-embedded (FFPE) blocks from multiple patients were visualized using TMA method to reduce the amount of reagents required for immunostaining of an entire array. Briefly, all FFPE tissue blocks were punched with a hollow needle and five tissue cores (3-mm wide) were removed. These samples were transferred into a positional encoded array in a paraffin block recipient that served as a new TMA. Tumor samples selected in this study were distributed in 12 TMAs (4-mm tissue cores), and sections from these TMAs were cut using a microtome, mounted on a microscope slide, and analyzed by immunohistochemistry. A normal breast tissue was included as an internal control in each TMA⁽²³⁾.

Immunohistochemical staining

Immunohistochemistry (IHC) for hormonal receptors as well as HER2 was performed during diagnosis using the streptavidin-biotin-peroxidase method. Samples were considered positive for estrogen and progesterone receptors when a weak, moderate, or strong nuclear staining was detected in more than 1% of the cells⁽²⁴⁾. HER2 status was evaluated according to Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 guidelines⁽²⁵⁾, which classify tumor into four categories (0, 1+, 2+, 3+) based on the percentage of positive cells and intensity of membrane staining. Cases interpreted as 3+ were considered positive, whereas 2+ tumors were considered equivocal and referred to fluorescent in situ hybridization (FISH). Cases interpreted as 0 or 1+ were considered negative.

IHC staining for CD44, CD44v6, and CD24 was also performed with the streptavidin-biotin-peroxidase method in 12 TMA slices (Table 2). Staining for CD44 was conducted at the Pathology

Laboratory of A.C. Camargo Cancer Center (São Paulo), while that for CD24 and CD44v6 was carried out at the Experimental Pathology Laboratory of Pontifícia Universidade Católica do Paraná (PUCPR). The expression of CD44, CD44v6, and CD24 was considered positive when the membranous or cytoplasmic immunostaining was ≥ 5% (Figure)⁽²⁶⁾. Samples detected positive for CD44 or CD44v6 and completely negative for CD24 were considered to display CD44⁺/ CD24^– phenotype. The slides were simultaneously analyzed by a researcher and a pathologist using a multi-observer microscope, without any knowledge of the clinical case.

Figure − IHC presentation of negative control (left panel) and positive (right panel) tissue for selected markers (40× magnification) IHC: immunohistochemistry.

Clinicopathological parameters

The age of patients was analyzed as a continuous variable by arithmetic mean and standard deviation. The histological grade was classified according to the Nottingham-Bloom-Richardson grading system⁽²⁷⁾. Tumor size was reported according to tumor-node-metastasis (TNM) (7^th edition, 2009) classification of the American Joint Committee in Cancer⁽²⁸⁾. Lymph node status was considered positive or negative according to the presence or absence of tumor cells in at least one lymph node. Invasive ductal carcinoma was defined according to the World Health Organization (WHO) Classification of Tumours of the Breast⁽²⁹⁾. The sample was classified into four molecular subtypes based on clinicopathological criteria:

• luminal − estrogen receptor (ER)+ and/or progesterone receptor (PR)+ and HER2-;

• luminal B-HER2 − ER+ and/or PR+ and HER2+;

• HER2 − ER−, PR-, and HER2+;

• triple negative (TN) − ER-, PR-, and HER2-.

Statistical analysis

The Student’s t-test for independent samples or analysis of variance (Anova) model was used to compare age between groups. Chi-square or Fisher’s exact test was used to evaluate the association between qualitative variables. Logistic regression model was used for multiple comparisons of qualitative variables between more than two groups and to evaluate the subtypes based on CD44⁺/CD24^– phenotype (adjusting for age, histological grade, and tumor size). A value of p < 0.05 was considered statistically significant. Data were analyzed with the software Statistica v.8.0.

Ethical aspects

This study was approved by the Ethics Committee of PUCPR, registration number 5365.

RESULTS

Characteristics of the sample

The study comprised 133 cases:

• 42 cases of luminal (ER+ and/or PR+ and HER2-);

• 31 cases of luminal B-HER2 (ER+ and/or PR+ and HER2+);

• 21 cases of HER2 (ER-, PR-, and HER2+);

• 39 cases of TN (ER-, PR-, and HER2-).

The mean and median ages were 55.8 years (range 27-88 years, standard deviation 13.8) and 54 years, respectively. The sample distribution in relation to histological grade, tumor size, and lymph node is described in Table 3.

Clinicopathological characteristics and molecular subtypes

Younger patients tended to harbor the TN subtype (p = 0.028). Of note, patients with TN BC had the lowest mean age. Individual comparison between groups showed that the average age of the TN cohort was significantly lower than that of the luminal phenotype (p = 0.008) and HER2+ (p = 0.019) cohort. Patients with TN BC and HER2+ disease had more aggressive conventional pathologic features. In comparison with luminal and luminal B-HER2 groups, the TN BC group had more grade III tumors (p < 0.001). Similar observation was reported for the HER2+ subgroup (Table 4).

Clinicopathological characteristics and CD44, CD44v6, and CD24 expression

The individual analysis of CD44, CD44v6, and CD24 resulted in the exclusion of four cases from CD24 group. In addition, four cases were excluded from the CD44v6 group; two lacked residual tumor on the slide and two histological sections had detached from the slide. One case was excluded from CD44 analysis because the histological section had detached from the slide. In 132 cases, 50% were positive for CD44 (66 cases). Out of 129 cases, 88 cases (68.2%) were positive for CD44v6, and 103 cases (79.8%) were positive for CD24. No significant association was observed between the expression of CD44, CD44v6, and CD24 and clinicopathological characteristics (Table 5).

Molecular subtypes, CD44⁺/CD24^– phenotype, and CD44, CD44v6, and CD24 expression

Eighteen of the 129 (14%) samples were positive for CD44 and/or CD44v6 expression and negative for CD24 expression. Among patients with CD44⁺/CD24^– phenotype, 13 (72%) were reported positive for CD44v6 expression and one (5.5%) was reported positive for CD44 expression; four (22.2%) patients showed expression of both markers. The evaluation of the expression of CD44⁺/CD24^– phenotype in different BC subtypes revealed a significant difference between groups (p = 0.02); the highest frequency was reported for HER2+ group. Comparison of the subtypes in pairs showed that HER2+ group demonstrated a higher frequency of CD44⁺/CD24^– phenotype as compared to luminal (p = 0.008) and TN (p = 0.044) group. Multivariate analysis revealed a higher frequency of CD44⁺/CD24^– phenotype in HER2+ subtype than luminal (p = 0.016) and TN (p = 0.050) subtype (Table 6). No significant difference was observed in CD44 expression between groups (Table 6). Comparison of CD44v6 expression showed a tendency to be statistically significant (p = 0.078) between the groups and luminal B-HER2 and HER2+ subtypes had the highest expression (Table 6). The expression of CD24 was statistically (p = 0.044) correlated to BC subtypes, with HER2+ subtype showing the lowest proportion of positive cases (Table 6).

DISCUSSION

The most significant finding of this study was the association between CD44⁺/CD24^– phenotype and BC molecular subtypes (p = 0.02); HER2+ subtype is known for poor prognosis and showed the highest frequency. Such association has been reported for the first time. HER2 subtype has been previously related to another stem-cell marker, aldehyde dehydrogenase 1 (ALDH1)^(30-33). CD44⁺/CD24^– phenotype may be associated with BC subgroups having a more primitive cellular origin (such as basal and claudin-low subtypes). Although several studies have shown this association^{(32, 34-36)}, this statement remains debatable⁽³⁷⁾. Honeth et al. (2011)⁽³⁵⁾ studied 240 tumors from a cohort of 445 patients surgically treated for stage II BC and found that the occurrence of CD44⁺/CD24^– phenotype was enriched in basal tumors, especially in hereditary BRCA1 tumors. Furthermore, the higher frequency of CD44⁺/CD24^– cells was associated with the basal subtype⁽³²⁾. Ricardo et al. (2011)⁽³⁶⁾ analyzed 466 cases of invasive mammary carcinomas and eight BC cell lines and observed a statistically significant correlation between CD44⁺/CD24^– phenotype and molecular subtypes of BC; CD44⁺/CD24^– phenotype was reported to be most frequent in basal tumors. In addition, Giatromanolaki et al. (2011)⁽³⁴⁾ described an association between CD44⁺/CD24^– phenotype and TN tumors. On the other hand, Lu et al. (2011)⁽³⁷⁾ suggested that CD44⁺/CD24^– cells may act as progenitor cells in transit and have no relation with molecular subtypes or clinical and pathological parameters of invasive ductal carcinoma.

To address the relationship between HER2 activation and stimulation of BC stem cells, Wang et al. (2010)⁽³⁸⁾ compared the phenotypic differences between two cell lineages derived from a common mammary epithelial stem cell with different HER2 expression. Flow cytometry demonstrated a higher frequency of CD44⁺/CD24^– cells in the HER2+ cell line, suggesting that HER2 displays the ability to increase the proportion of CD44⁺/CD24^– stem cells. Korkaya et al. (2008)⁽³⁹⁾ showed that HER2 overexpression increases the proportion of stem/progenitor cells, in normal mammary epithelial cells by using in vitro mammosphere assays, and the expression of stem-cell marker ALDH. The author also demonstrated that HER2 overexpression increases and HER2 blockade decreases the cancer stem-cell population in breast cancer cell lines and mouse xenografts.

The development of HER2-targeting agents such as trastuzumab has substantially changed the HER2-overexpressed tumors prognosis in the latest years, especially when used in the neoadjuvant setting⁽⁴⁰⁾. Recent data have shown that probably the great results of these agents are related to its ability to target the breast cancer stem-cell population⁽⁴¹⁾. Traditionally these agents have been indicated only for tumors that overexpress HER2, but Paik et al. (2008)⁽⁴²⁾ reanalyzed clinical samples accrued to the National Surgical Adjuvant Breast and Bowel Project protocol B-31 (NSABP B31), for HER2 gene amplification, and identified 174 cases which were originally reported as HER2+ but did not actually have HER2 gene amplification. Surprisingly, these patients had the same benefit of trastuzumab as the patients that were HER2 positive. One of the hypotheses to explain this finding is that the HER2 pathway is activated in the cancer stem-cell populations of this group of patients⁽⁴¹⁾. To answer this question, a randomized prospective phase III trial, NSABP B47, is in progress to determine the benefits of adjuvant trastuzumab in patients that are not HER2 positive⁽⁴¹⁾.

In the development of new anti-tumor drugs, targeting the cancer stem-cell populations can provide new strategies for breast cancer treatment. Bozepinib is one of the most active compound of new (RS)-4,1-benzoxazepin-purines against human breast and colon cancer cell lines. It shows very low inhibitory concentration 50 (IC50) values, in both breast and colon cancer cells, and induces cell death by apoptosis⁽⁴³⁾. Ramirez et al. (2014)⁽⁴⁴⁾ further studied the mechanism of action of bozepinib and found that this agent is a selective inhibitor of HER2 positive breast cancer cells. Moreover, the authors showed that bozepinib inhibited both mammo- and colonosphere formation in subpopulations isolated by ALDH activity, and was able to induce apoptosis in the resistant ALDH+ subpopulations, presenting promising data about the action of this agent over cancer stem cells⁽⁴⁴⁾.

Cancer stem cells are commonly related to the mechanism of chemotherapy and radiotherapy resistance⁽¹⁶⁾. Tumors overexpressing stem-cell markers were expected to be associated with poor prognosis and clinicopathological characteristics of more aggressive behavior. However, the relationship between cancer stem-cell marker and prognosis remains controversial. The phenotype CD44⁺/CD24^– was associated with more aggressive clinicopathological features and, consequently, poor prognosis^{(34, 45)}, although some contradictory studies have reported poor prognosis in BCs enriched with CD44-/CD24+⁽⁴⁶⁾. It was recently demonstrated that cancer stem-cell markers may be identified in tumors with worst prognosis within the basal subtype characterized with a higher expression of CD44⁺/CD24^– phenotype⁽⁴⁷⁾. We failed to observe any relationship between CD44⁺/CD24^– phenotype and the classic prognostic factors as age, histological grade, or lymph node status, as previously described in other studies^{(35, 37, 48, 49)}.

A major limitation of this study is that we performed simple immunostaining to characterize CD44⁺/CD24^– population. Ricardo et al. (2011)⁽³⁶⁾ conducted a comparative study between simple immunostaining and double immunostaining with fluorescence microscopy using the same anti-CD44 and anti-CD24 in both TMA and whole tissue and found similar results with both techniques. Our study included IHC staining of CD44v6 to assess CD44⁺/CD24^– phenotype. CD44 is encoded by a single gene located on 11p13 chromosome. The standard isoform (CD44s) is the most common, but approximately 20 variants of CD44 formed by alternative splicing have been described. CD44v6 is one of the most common and best studied CD44 variants known to play a role in cell migration and proliferation and, consequently, cancer progression and metastasis^{(50, 51)}. In addition, it is associated with aggressive tumor behavior and poor prognosis⁽⁵²⁾. Although our results have shown no statistical association between presence of CD44v6 and cancer subtypes, we observed a tendency of association between the expression of CD44v6 and the luminal B-HER2 and HER2+ subtypes. We also revealed higher rates of CD24 with cytoplasmic immunostaining (79.8%). A significant association was also demonstrated between CD24 negativity and BC molecular subtypes; HER2 was the most frequent subtype in this group, and the correlation between BC subtypes and CD24 revealed the lowest expression of CD24 in HER2+ subtype.

CONCLUSION

Our results demonstrated an association between the CD44⁺/CD24^– phenotype and molecular subtypes, with the highest expression observed in HER2 subtype. Cancer stem cells are possibly responsible for the mechanism of resistance to BC treatment, owing to their ability to survive chemotherapy and radiotherapy. Identification of these cells through cancer stem-cell markers may possibly open new pathways for BC treatment.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

We would like to thank Mrs. Marina de Azevedo and Ms. Ana Paula Camargo for their technical assistance, Dr. Rafael Malagoli Rocha (A.C. Camargo Pathology Laboratory) for carrying out CD44 IHC, and Dr. Marcia Olandoski for statistical analysis and for her support with tables and figures.

REFERENCES

1. Parkin DM, Bray F, Ferlay J, Pisani P Estimating the world cancer burden: Globocan 2000. Int J Cancer. 2001; 94(2): 153-6.

2. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol. 2006; 24(14): 2137-50.

3. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature. 2000; 406(6797): 747-52.

4. Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001; 98(19): 10869-74.

5. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003; 100(14): 8418-23.

6. Sorlie T. Molecular portraits of breast cancer: tumour subtypes as distinct disease entities. Eur J Cancer. 2004; 40(18): 2667-75.

7. Parker JS, Mullins M, Cheang MC, et al. Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol. 2009; 27(8): 1160-7.

8. Prat A, Parker JS, Karginova O, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 2010; 12(5): R68.

9. Ihemelandu CU, Leffall Jr. LD, Dewitty RL, et al. Molecular breast cancer subtypes in premenopausal and postmenopausal African-American women: age-specific prevalence and survival. J Surg Res. 2007; 143(1): 109-18.

10. Millikan RC, Newman B, Tse CK, et al. Epidemiology of basal-like breast cancer. Breast Cancer Res Treat. 2008; 109(1): 123-39.

11. Carey LA, Perou CM, Livasy CA, et al. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA. 2006; 295(21): 2492-502.

12. Goldhirsch A, Wood WC, Coates AS, et al. Strategies for subtypes–dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol. 2011; 22(8): 1736-47.

13. Prat A, Pineda E, Adamo B, et al. Clinical implications of the intrinsic molecular subtypes of breast cancer. Breast. 2015; 24 Suppl 2: S26-35.

14. Graziano A, d’Aquino R, Tirino V, Desiderio V, Rossi A, Pirozzi G. The stem cell hypothesis in head and neck cancer. J Cell Biochem. 2008; 103(2): 408-12.

15. Kakarala M, Wicha MS. Implications of the cancer stem-cell hypothesis for breast cancer prevention and therapy. J Clin Oncol. 2008; 26(17): 2813-20.

16. Tang C, Chua CL, Ang BT. Insights into the cancer stem cell model of glioma tumorigenesis. Ann Acad Med Singapore. 2007; 36(5): 352-7.

17. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003; 100(7): 3983-8.

18. Baumann P, Cremers N, Kroese F, et al. CD24 expression causes the acquisition of multiple cellular properties associated with tumor growth and metastasis. Cancer Res. 2005; 65(23): 10783-93.

19. Schabath H, Runz S, Joumaa S, Altevogt P. CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells. J Cell Sci. 2006; 119(Pt2): 314-25.

20. Sheridan C, Kishimoto H, Fuchs RK, et al. CD44⁺/CD24^– breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res. 2006; 8(5): R59-

21. Fillmore CM, Kuperwasser C. Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res. 2008; 10(2): R25.

22. Phillips TM, McBride WH, Pajonk F. The response of CD24(-/low)/ CD44⁺ breast cancer-initiating cells to radiation. J Natl Cancer Inst. 2006; 98(24): 1777-85.

23. Chong DC, Raboni SM, Abujamra KB, et al. Respiratory viruses in pediatric necropsies: an immunohistochemical study. Pediatr Dev Pathol. 2009; 12(3): 211-6.

24. Hammond ME, Hayes DF, Wolff AC, Mangu PB, Temin S. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Oncol Pract, 2010; 6(4): 195-7.

25. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014; 138(2): 241-56.

26. Tsang JY, Huang YH, Luo MH, et al. Cancer stem cell markers are associated with adverse biomarker profiles and molecular subtypes of breast cancer. Breast Cancer Res Treat. 2012; 136(2): 407-17.

27. Elston CW. Classification and grading of invasive breast carcinoma. Verh Dtsch Ges Pathol. 2005; 89: 35-44.

28. Sobin LH, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours. 7^th ed. Wiley-Blackwell; 2009. 336 p.

29. Lakhani SR, Ellis IO, Schnitt SJ, Tan PH, van de Vijver MJ. WHO classification of tumours of the breast. Lyon: International Agency for Research on Cancer (IARC); 2012.

30. Ginestier C, Hur MH, Charafe-Jauffret E, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007; 1(5): 555-67.

31. Morimoto K, Kim SJ, Tanei T, et al. Stem cell marker aldehyde dehydrogenase 1-positive breast cancers are characterized by negative estrogen receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression. Cancer Sci. 2009; 100(6): 1062-8.

32. Park SY, Lee HE, Li H, Shipitsin M, Gelman R, Polyak K. Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer. Clin Cancer Res. 2010; 16(3): 876-87.

33. Zhou L, Jiang Y, Yan T, et al. The prognostic role of cancer stem cells in breast cancer: a meta-analysis of published literatures. Breast Cancer Res Treat. 2010; 122(3): 795-801.

34. Giatromanolaki A, Sivridis E, Fiska A, Koukourakis MI. The CD44⁺/CD24^– phenotype relates to ‘triple-negative’ state and unfavorable prognosis in breast cancer patients. Med Oncol. 2011; 28(3): 745-52.

35. Honeth G, Bendahl PO, Ringner M, et al. The CD44⁺/CD24^– phenotype is enriched in basal-like breast tumors. Breast Cancer Res. 2008; 10(3): R53.

36. Ricardo S, Vieira AF, Gerhard R, et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol. 2011.

37. Lu X, Xu K, Lu H, et al. CD44(+)/CD24(-) cells are transit progenitors and do not determine the molecular subtypes and clinical parameters in breast carcinomas. Ultrastruct Pathol. 2011; 35(2): 72-8.

38. Wang KH, Kao AP, Chang CC, et al. Increasing CD44⁺/CD24(-) tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis. Mol Cancer. 2010; 9: 288.

39. Korkaya H, Paulson A, Iovino F, Wicha MS. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene. 2008; 27(47): 6120-30.

40. Buzdar AU, Ibrahim NK, Francis D, et al. Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer. J Clin Oncol. 2005; 23(16): 3676-85.

41. Korkaya H, Wicha MS. HER2 and breast cancer stem cells: more than meets the eye. Cancer Res. 2013; 73(12): 3489-93.

42. Paik S, Kim C, Wolmark N. HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med. 2008; 358(13): 1409-11.

43. Marchai JA, Carrasco E, Ramirez A, et al. Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNalpha triggering apoptosis, autophagy and senescence. Drug Des Devel Ther. 2013; 7: 1301-13.

44. Ramirez A, Boulaiz H, Morata-Tarifa C, et al. HER2-signaling pathway, JNK and ERKs kinases, and cancer stem-like cells are targets of Bozepinib small compound. Oncotarget. 2014; 5(11): 3590-606.

45. Idowu MO, Kmieciak M, Dumur C, et al. CD44(+)/CD24(-/low) cancer stem/progenitor cells are more abundant in triple-negative invasive breast carcinoma phenotype and are associated with poor outcome. Hum Pathol. 2012; 43(3): 364-73.

46. Ahmed MA, Aleskandarany MA, Rakha EA, et al. A CD44(-)/CD24(+) phenotype is a poor prognostic marker in early invasive breast cancer. Breast Cancer Res Treat. 2012; 133(3): 979-95.

47. Chekhun SV, Zadvorny TV, Tymovska YO, Anikusko MF, Novak OE, Polishchuk LZ. CD44⁺/CD24^– markers of cancer stem cells in patients with breast cancer of different molecular subtypes. Exp Oncol. 2015; 37(1): 58-63.

48. Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, Brauch H. Prevalence of CD44⁺/CD24^–/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin Cancer Res. 2005; 11(3): 1154-9.

49. Mylona E, Giannopoulou I, Fasomytakis E, et al. The clinicopathologic and prognostic significance of CD44⁺/CD24(-/low) and CD44-/CD24+ tumor cells in invasive breast carcinomas. Hum Pathol. 2008; 39(7):1096-102.

50. Gunthert U, Hofmann M, Rudy W, et al. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell. 1991; 65(1): 13-24.

51. Sneath RJ, Mangham DC. The normal structure and function of CD44 and its role in neoplasia. Mol Pathol. 1998; 51(4): 191-200.

52. Schmitt F, Ricardo S, Vieira AF, Dionisio MR, Paredes J. Cancer stem cell markers in breast neoplasias: their relevance and distribution in distinct molecular subtypes. Virchows Arch. 2012; 460(6): 545-53.