Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer

Abstract

Purpose: To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage.

Experimental design: We performed immunohistochemical analyses of 12 proteins [CD44, CD24, ALDH1, vimentin, osteonectin, EPCR, caveolin 1, connexin 43, cytokeratin 18 (CK18), MUC1, claudin 7, and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only, 135 cases of IDC with ductal carcinoma in situ (DCIS), 35 cases of DCIS with microinvasion, and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry.

Results: CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors, especially in luminal A subtype. The expression of vimentin, osteonectin, connexin 43, ALDH1, CK18, GATA3, and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells.

Conclusions: Our findings suggest that in breast cancer, the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage.

Figure 1. Immunohistochemical analyses of CD44, CD24, and ALDH1 in breast cancer

A, Immunohistochemical staining patterns in normal breast tissue. CD24 and CD44 displayed uniform luminal and basal cell-specific pattern, respectively, whereas ALDH1 expression varied depending on the area of the tissue. B, Representative examples of immunohistochemical analyses of invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS) according to tumor subtype using the indicated antibodies. In luminal A subtype tumor cells positive for CD44 and CD24 (apical membranous pattern) can be seen whereas ALDH1 was not detected. HER2+ tumor is negative for CD44, and positive for CD24 (membrano-cytoplasmic pattern) and ALDH1. In basal-like subtype tumor cells are positive for CD44 and ALDH1, but negative for CD24. C, Representative examples of double immunohistochemistry for CD44 (brown) and CD24 (red) in IDC and in DCIS.

Figure 2. Immunohistochemical analyses of genes differentially expressed between CD44+ and CD24+ breast cancer cells

A, Representative examples of immunohistochemical staining patterns observed in invasive breast tumors using antibodies against the indicated proteins. A subset of markers that were highly expressed in CD44+ compared to CD24+ breast cancer cells based on our prior study are expressed only in basal-like tumors (vimentin, osteonectin, and caveolin 1), whereas Connexin 43 is expressed both in luminal A and basal-like subtypes. Markers more abundantly expressed in CD24+ compared to CD44+ breast cancer cells (Keratin 18, MUC1, and GATA3) are expressed in luminal A tumors. B, Representative examples of immunohistochemical staining patterns observed in DCIS using antibodies against the indicated proteins. Vimentin is expressed in basal-like but not in luminal A DCIS. Strong vimentin staining is detected in stromal cells in both tumor subtypes. In contrast, GATA3 is expressed in luminal A but not in basal-like DCIS. C, Immunohistochemical analyses of the expression of markers between invasive (red star) and in situ (black star) components of the same tumor. CD44 is expressed in both invasive and in situ components of luminal A subtype. HER2+ tumors are negative for CD44, only stromal staining is detected. In basal-like IDC keratin 18 is expressed only in the in situ component whereas vimentin is expressed in both invasive and in situ areas.