The Differential Expression of Cancer Stem Cell Markers CD<sub>44</sub>, CD<sub>24</sub> and ALDH1 in Breast Cancer Histological Types

Research Article

Austin J Surg. 2015; 2(6): 1075.

The Differential Expression of Cancer Stem Cell Markers CD44, CD24 and ALDH1 in Breast Cancer Histological Types

Lee JS¹, Kim YM²* and Kim WK²*

¹Department of Surgery, Haeundae Paik Hospital, Inje University, South Korea

²Department of Pathology, Haeundae Paik Hospital, Inje University, South Korea

*Corresponding author: Kim WK and Kim YM, Department of Pathology, Haeundae Paik Hospital, Inje University College of Medicine, 875 Haeun-daero, Haeundae-gu, Busan 612-030, South Korea

Received: July 29, 2015; Accepted: November 06, 2015; Published: November 17, 2015


Aldehyde Dehydrogenase 1(ALDH-1) and CD44+CD24- are the most consistently used biomarkers to identify and characterize breast Cancer Stem Cells (CSCs). However, expression of CSCs in Specific Histologic Types (SHTs) of Invasive Ductal Carcinomas (IDCs) remains unclear. We aimed to determine the distribution of CD44, CD24 and ALDH-1 breast CSC markers in SHTs of IDC compared with that in IDC-Not Otherwise Specified (NOS). From October 2013 to February 2014, we analyzed 168 IDC cases for double immunohistochemical staining of CD44, CD24 and single expression of ALDH- 1. The distribution of these CSC markers, CD44+CD24-, ALDH-1 and CSC phenotypes (CD44+CD24-/ ALDH-1) were retrospectively evaluated among the distinct SHTs and intrinsic subtypes compared with IDC-NOS. Medullary, metaplastic and apocrine carcinomas enriched in ALDH-1 population (100%, 100%, and 66.7%, respectively, vs. 44.1% in IDC-NOS, P=0.04). Medullary, papillary, and metaplastic carcinomas displayed significant increases in CSC phenotype frequency (66.7%, 28.6% and 100%, respectively, vs. 20% in IDCNOS, P=0.03). In IDC-SHT, mucinous carcinoma was correlated with luminal A subtype and medullary, papillary, metaplastic, apocrine, and micropapillary carcinomas were correlated with triple negative breast cancer (P=0.01). CK5/6(+), EGFR(+), ER(-), PR(-), and high HG were associated with ALDH-1(+) or CSC phenotype, but HER2(-) was associated with CD44+CD24-(+). Within distinct SHTs, medullary and metaplastic carcinomas are highly associated with the basal-like subtype and the CSC phenotype. In conclusion, we demonstrated the described CD44+/CD24-, ALDH-1, and CSC phenotypes may identify CSCs with distinct levels of differentiation and several SHTs are distinguished entities from IDC-NOS with regard to CSC marker expression.

Keywords: Breast cancer; Cancer stem cell; Basal-like; Intrinsic subtype


ALDH-1: Aldehyde Dehydrogenase-1; CSC: Cancer Stem Cell; SHT: Specific Histologic Type; IDC: Invasive Ductal Carcinoma; NOS: Not Otherwise Specified; CK5/6(+): Cytokeratin 5/6; EGFR: Epidermal Growth Factor Receptor; ER: Estrogen Receptor; PR: Progesterone Receptor; HG: Histologic Grade; HER2: her-2/neu over expressed; TNBC: Triple Negative Breast Cancer


Breast cancer is a heterogeneous disease, comprising various histological types, with distinct clinical presentations and underlying molecular signatures. Despite an increased knowledge about breast cancers in clinicians, development of metastasis cannot be always avoided in patients. One explanation for treatment failure is the Cancer Stem Cell (CSC) theory, which hypothesizes that cancer, may originate from and be sustained by a small population of cells that display the ability to maintain tumor growth by self-renewal and differentiation, as well as resistance to chemotherapy and radiotherapy [1-3]. In the case of breast tumors, Al-Hajj et al. [4] were the first to isolate a highly tumorigenic subpopulation of tumor cells with a CD44+CD24- lineage phenotype. Subsequently, Ginestier et al. [5] presented evidence that Aldehyde Dehydrogenase-1 (ALDH- 1) is a marker of stem/progenitor cells of the normal and malignant human breast. Based on this current knowledge, there is evidence to support the idea that the use of CD44 and CD24 cell surface markers in combination with ALDH-1 activity is the most accurate method to identify and isolate CSC-like cells within breast cancer populations. However, none of these CSC markers is expressed exclusively by stem cells, and a considerable number of cells that express these markers are not stem cells, resulting in phenotype heterogeneity within putative CSC populations [6,7]. Moreover, the overlap between high CD44+CD24-(+) and ALDH-1 expressions in primary tumors is quite small (approximately 1%) [5], thus it is imperative to improve CSC identification in routine formalin-fixed and paraffin-embedded tissue samples. Furthermore, regarding Special Histological Types (SHTs) that comprise up to 25% of invasive breast cancers [8], only a few studies have been conducted to explore the role of CD44 and CD24 in Micro papillary Carcinomas of the breast (IMPC) [9]. In a recent study by Park et al. [10], several stem cell-related markers were tested, but only Invasive Ductal Carcinomas (IDC)-Not Otherwise Specified (NOS) cases were studied. Others used cohorts mainly composed of IDC-NOS samples with only few cases of SHT [11,12]. Therefore, the frequency of the CSC phenotype in SHT breast carcinomas and whether each SHT has its own CSC remains largely unknown. In present study, we analyzed the expression of the main established breast CSC markers, CD44, CD24, and ALDH-1, in a large series of IDCs to evaluate their distribution among the different intrinsic subtypes and SHTs.

Methods and Materials

Patient selection

We prospectively collected 188 primary breast cancer specimens between October 2013 and February 2014 at our institute through our breast cancer center. One hundred sixty eight breast cancer patients were included for this study after exclusions. The following exclusion criteria for breast cancer patients or healthy subjects were applied in this study: (a) neoadjuvant chemotherapy (n=2); (b) recurrent breast cancer (n=1); (c) bilateral breast cancer (n=5); (d) ductal carcinoma in situ (n=7); (e) invasive lobular carcinoma (n=4); and (f) lobular carcinoma in situ (n=1). All patients provided written informed consents, and use of biological specimens, as well as clinical data for research purposes, were approved by the Institutional Review Boards of Haeundae-Paik Hospital, Inje University (Haeundae-paik 2013- 60).

Tissue specimens

Tissue Microarrays (TMA) were constructed from representative tissue columns (2.0mm in diameter) of formalin-fixed paraffin embedded tissue. For each surgical case, two cores from every individual tumor were made into TMA. All patients were female, with a median age of 52.3±10.2 years. Clinicopathological information was obtained by reviewing medical records, pathological reports and hematoxylin and eosin stained slides, and included the followings: tumor size, ipsilateral axillary lymph node status, histologic subtype, Bloom–Richardson histologic grade, lymphovascular tumor emboli, Estrogen Receptor (ER) status, Progesterone Receptor (PR) status, Human Epidermal Growth Factor Receptor 2 (HER2) status, and Ki67 index, as well as expressions of basal cell markers, Epidermal Growth Factor Receptor (EGFR) and cytokeratin 5/6.

Immunohistochemical staining of CD44 and CD24

Sections (4μm thick) of the TMA blocks were mounted on Superfast Plus Slides (Thermo Scientific). Double-immunostaining was performed using a detection kit (Ventanaultraview universal kit) by a Ventana Benchmark XT Autostainer (Ventana medical system). CD44 staining (1:200 dilution; Clone 156-3C11, Neomarkers, Fremont, CA, USA) was visualized with AP Red (Ventanaultraview universal AP red kit), whereas CD24 (1:50 dilution; polyclonal CD24 antibody, Biorbyt, Cambridge, UK) was visualized using Diaminobenzidine (DAB) (Ventanaultraview universal DAB kit).

Immunohistochemical staining of ALDH-1

For ALDH-1 immunostaining, ALDH-1 antibody (1:100 dilution; clone 44/ALDH, BD Biosciences, San Jose, CA, USA) was used. Staining was performed using the Ventanaultraview universal DAB kit according to the manufacturer’s protocols. Paraffin sections of normal liver tissue were used as a positive control. The cytoplasmic staining of cancer cells was considered ALDH-1 positive. Positive control for the specificity of ALDH-1 staining was carried out in normal hepatic tissue showing diffuse strong cytoplasmic expression.

Immunohistochemical scoring

All cases were independently reviewed and scored by two pathologists (W.G. Kim and Y.M. Kim) who were blinded to clinical diagnosis and there was inter-pathologist discussion for all unclear cases.

CD44 showed predominant membranous staining patterns and the scoring was carried out as follows: 0, 0% positive tumor cells; 1, 1–10% positive cells; 2, 11–50% positive cells; 3, 51–75% positive cells; and 4, 76–100% positive cells. Conversely, CD24 staining was detected mainly in the cytoplasm with some membranous staining pattern. The same scoring system described for CD44 was applied for CD24.

The proportion of CD44+CD24-(+) tumor cells was determined as the percentage of cells positive for AP Red staining but negative for DAB staining (Figure 1A-1D). For subsequent analyses, tumors with score of 2 to 4 (>10% staining) were considered positive for CD44+CD24- phenotype. The frequencies of CD44-CD24+ cells, CD44+CD24+ cells and CD44-CD24- tumor cells were determined in a similar fashion.