Molecular characterization of CD44+/CD24-/Ck+/CD45- cells in benign and malignant breast lesions.

Breast cancer epithelial cells with the CD44+/CD24-/low phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24-/cytokeratin(Ck)+/CD45- cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24-/Ck+/CD45- cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24-/Ck+/CD45- cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24-/Ck+/CD45- cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.

Characterization of CD44+ALDH1+Ki-67- Cells in Non-malignant and Neoplastic Lesions of the Breast.

BACKGROUND: Cancer stem cells are tumor cells that present self-renewal, clonal tumor initiation capacity and clonal long-term repopulation potential. We have previously demonstrated that the co-expression of the breast cancer stem cell (BCSC) markers hyaluronan receptor (CD44) and aldehyde dehydrogenase-1 (ALDH1) in ductal carcinomas in situ could be determinant for disease progression. Combining these established BCSC markers with Ki-67 to evaluate quiescence we sought to identify, evaluate the distribution and estimate the mean percentages of CD44(+)ALDH1(+)Ki-67(-) breast cells. MATERIALS AND METHODS: Triple-immunohistochemistry for CD44, ALDH1 and Ki-67 was applied in a series of 16 normal, 54 non-malignant and 155 malignant breast tissues. Clinical relevance was inferred by associations with markers of breast cancer behavior, progression and survival. RESULTS: The mean percentages of cells with this phenotype increased significantly from non-malignant lesions to high-grade ductal carcinomas in situ, decreasing in invasive ductal carcinomas, as also evidenced by an inverse correlation with histological grade and tumor size. The mean percentage of CD44(+)ALDH1(+)Ki-67(-) cells was also significantly higher in women who developed distant metastasis and died due to breast cancer, and a significant association with human epidermal growth factor type 2 (HER2) negativity was observed. CONCLUSION: Our novel findings indicate that CD44(+)ALDH1(+)Ki-67(-) tumor cells may favor distant metastasis and can predict overall survival in patients with ductal carcinomas of the breast. More importantly, quiescence may have a crucial role for tumor progression, treatment resistance and metastatic ability of BCSCs.

Co-expression of stem cell markers ALDH1 and CD44 in non-malignant and neoplastic lesions of the breast.

BACKGROUND/AIM: The Cancer Stem Cell (CSC) model proposes that cancer is driven by a cellular component which possesses stem cell (SC) properties, cancer stem cells (CSCs), a distinct cell-type which is tumorigenic and capable of invasion and metastasis. Enzymatic activity of aldehyde dehydrogenase-1 (ALDH1), a de-toxifying enzyme that oxidizes intracellular aldehydes, has been used as a marker of normal and malignant breast stem cells (BSCs). CD44-transmembrane protein has already been shown to possess the ability to identify breast epithelial cells with stem properties. MATERIALS AND METHODS: In order to compare two of the currently most reliable BSCs markers, ALDH1 and CD44, and to correlate their expression within different breast lesions, 190 samples from breast cancer specimens were analyzed by tissue microarrays. RESULTS: ALDH1 expression was observed in 85.43% and CD44 in 90.3% of all samples. No overexpression was observed for ALDH1 between invasive tumors, ductal carcinomas in situ and non-malignant lesions of breast, although ALDH1 had a significant negative correlation with estrogen-receptor (ER) and progesterone-receptor (PR) status (p=0.002 and p=0.001, respectively) and a positive correlation with CD44 (p<0.001). Moreover, combined overexpression of ALDH1 and CD44 was observed in ductal in situ tumors (p<0.001). CONCLUSION: The combined overexpression of these markers in ductal carcinomas in situ is in agreement with the CSC model in breast cancer.