Patients and specimens
This study included 133 invasive ductal carcinomas, the most common breast cancer type, obtained from the archives of the Department of Pathology, Farhat Hached University Hospital of Sousse (Tunisia).
Different clinical and histological parameters including patient age, menopausal status, tumor size, lymph node metastasis, histological grade, hormone receptor and HER2 status, were collected from the Central Cancer Registry. Follow-up data were available for 74 patients. The overall follow-up time ranged from 4 to 143 months, with a median follow-up of 48 months, during which 11 patients underwent cancer relapse and 6 died.
The slides were reviewed and the cases were classified into 4 categories based on the immunohistochemical status of estrogen receptors (ER), progesterone receptors (PR), HER2 and Ki67, according to Goldhirsch et al. : luminal A (ER+ and/or PR+, HER2−, low Ki67), luminal B (ER+ and/or PR+, HER2+ and/or high Ki67), HER2 overexpressing (ER−, PR−, HER2+) and triple negative (ER−, PR−, HER2−).
Analysis of CD10 expression
The expression of CD10 (clone 56C6; dilution 1:100; Novocastra) was investigated by immunohistochemistry using the EnVision Flex system (DakoCytomation, Glostrup, Denmark) according to the manufacturer’s instructions.
Briefly, Paraffin-embedded breast cancer tissues was cut at 5 μm, dried overnight at 60 °C and deparaffinized in Ottix Plus (Diapath, Martinengo, Italy). Subsequently, the sections were hydrated with Ottix Shapper (Diapath, Martinengo, Italy), and rehydrated in water.
For antigen retrieval, the sections were boiled in a water bath with citrate buffer (0.01 M, pH 6.0) for 40 min until the temperature reached 98 °C. They were then allowed to cool at room temperature for 20 min, and placed in EnVision Flex Wash buffer (DakoCytomation, Glostrup, Denmark). The endogenous peroxidase activity was blocked with EnVision Flex Peroxidase-Blocking Reagent for 5 min. The sections were thoroughly washed with the Wash buffer. The samples were incubated 30 min at temperature room with the primary antibody. Subsequently, the sections were rinsed gently with Wash buffer.
The sections were stained using the high sensitive polymer-based EnVision Flex/HRP system. After being rinsed in wash buffer, they were incubated in 3, 3 diaminobenzidine a substrate–chromogen solution for 20 min. Finally, the slides were counterstained with Mayer hematoxylin, permanently mounted, and viewed with a standard light microscope. Positive and negative controls were included for antibody according to the kit instructions.
The immunostaining results were evaluated independently by two pathologists (M.T. and S.Z.). CD10 expression was evaluated in the neoplastic and stromal cells. A case was considered positive if more than 10% of the cells exhibited positive signal, otherwise it was negative [11, 20].
Analysis of cancer stem cell phenotype
The cancer stem cell phenotype was assessed by the immunohistochemical analysis of the expression of two cancer stem cell markers CD44 (clone DF1485, dilution 1:100, Leica, Newcastle, UK) and ALDH1 (clone 400 M-15, dilution 1:100, Cell Marque, Rocklin, California, USA). The antigen retrieval method and revelation system were the same as described for CD10.
Sequential double staining immunohistochemistry protocol was also used for CD10, ALDH1 and CD44 staining. We used the Bond Polymer Refine Red Detection (Fast Red) and the Bond Polymer Refine Detection (DAB) for the automated Bond system (Leica Microsystems, Wetzlar, Germany).
CD44 expression was evaluated in the cell membrane, whereas ALDH1 expression was evaluated in the cytoplasm of the tumor cells. For the two antibodies, a case was considered positive if more than 10% of the cells exhibited immunostaining, otherwise it was considered negative [10, 12, 21, 22].
Data analysis has performed using the SPSS software (version 20.0; SPSS, Chicago, IL, USA). The correlation between CD10 expression and the clinicopathological parameters was investigated by the Chi-square and the Fisher exact tests, where appropriate. The survival analyses were performed according to the Kaplan-Meier method and compared by the log-rank test. A p value ≤0.05 was considered to indicate statistical significance.