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

Abstract

Recently, aldehyde dehydrogenase (ALDH) 1 has been identified as a reliable marker for breast cancer stem cells. The aim of our study was to investigate the clinicopathological characteristics of breast cancers with ALDH1+ cancer stem cells. In addition, the distribution of ALDH1+ tumor cells was compared on a cell-by-cell basis with that of estrogen receptor (ER)+, Ki67+, or human epidermal growth factor receptor type 2 (HER2)+ tumor cells by means of double immunohistochemical staining. Immunohistochemical staining of ALDH1 was applied to 203 primary breast cancers, and the results were compared with various clinicopathological characteristics of breast cancers including tumor size, histological grade, lymph node metastases, lymphovascular invasion, ER, progesterone receptor, HER2, Ki67, and topoisomerase 2A as well as prognosis. Immunohistochemical double staining of ALDH1 and ER, Ki67, or HER2 was also carried out to investigate their distribution. Of the 203 breast cancers, 21 (10%) were found to be ALDH1+, and these cancers were significantly more likely to be ER- (P = 0.004), progesterone receptor- (P = 0.025), HER2+ (P = 0.001), Ki67+ (P < 0.001), and topoisomerase 2A+ tumors (P = 0.012). Immunohistochemical double staining studies showed that ALDH1+ tumor cells were more likely to be ER-, Ki67-, and HER2+ tumor cells. Patients with ALDH1 (score 3+) tumors showed a tendency (P = 0.056) toward a worse prognosis than did those with ALDH1- tumors. Breast cancers with ALDH1+ cancer stem cells posses biologically aggressive phenotypes that tend to have a poor prognosis, and ALDH1+ cancer stem cells are characterized by ER-, Ki67-, and HER2+.

Figure 1

Immunohistochemical identification of aldehyde dehydrogenase (ALDH) 1⁺ tumor cells. Representative results of immunostaining of ALDH1: 0 (negative), 1+ (weakly positive), 2+ (intermediately positive), and 3+ (strongly positive).

Figure 2

Relationship between hormone receptor (HR)/HER2 status and aldehyde dehydrogenase (ALDH) 1 or Ki67. Breast cancers were classified into four categories according to their HR and HER2 status: HR⁺ and HER2⁺, HR⁺ and HER2⁻, HR⁻ and HER2⁺, and HR⁻ and HER2⁻. The frequencies of (a) ALDH1⁺ and (b) Ki67⁺ tumors were compared.

Figure 3

Immunohistochemical double staining of aldehyde dehydrogenase (ALDH) 1 and Ki67. Representative results of immunohistochemical double staining of ALDH1 (red) and Ki67 (brown) in two breast cancers: (a–c) tumor #1, and (d) tumor #2. (a) ALDH1⁺ and ALDH1⁻ tumor cells were localized separately in tumor #1. Higher magnification of the ALDH1⁺ and ALDH1⁻ areas of this tumor revealed that (b) ALDH1⁺ tumor cells were mostly Ki67⁻ and (c) ALDH1⁻ tumor cells were more likely to be Ki67⁺. (d) In tumor #2, ALDH1⁺ tumor cells are localized in a mosaic pattern, and ALDH1⁺ tumor cells (black arrows) and Ki67⁺ tumor cells (white arrows) rarely overlapped.

Figure 4

Immunohistochemical double staining of aldehyde dehydrogenase (ALDH) 1 and estrogen receptor (ER). Representative results of immunohistochemical double staining of ALDH1 (red) and ER (brown) in two breast cancers: (a–c) tumor #3, and (d) tumor #4. (a) ALDH1⁺ tumor cell clusters and ALDH1⁻ tumor cell clusters were observed. (c) Higher magnification of an ALDH1⁺ tumor cell cluster revealed that ALDH1⁺ tumor cells were rarely ER⁺, and (b) that of an ALDH1⁻ tumor cell cluster revealed that ALDH1⁻ tumor cells were mostly ER⁺. (d) In tumor #4, ALDH1⁺ tumor cells were localized in a mosaic pattern, and ALDH1⁺ tumor cells (black arrows) and ER⁺ tumor cells (white arrows) rarely overlapped.

Figure 5

Immunohistochemical double staining of aldehyde dehydrogenase (ALDH) 1 and HER2. Representative results of immunohistochemical double staining of ALDH1 and HER2 in breast cancer (tumor #5) are shown. (a) HER2⁺ tumor cell clusters surrounded by HER2⁻ tumor cells were observed. (b) Higher magnification of an HER2⁺ tumor cell cluster revealed that ALDH1⁺ tumor cells (black arrows) were included in this cluster. (c) There was also a cluster not containing ALDH1⁺ tumor cells. (d) All HER2⁻ tumor cells were ALDH1⁻.

Figure 6

Comparison of aldehyde dehydrogenase (ALDH) 1⁺ tumor cells between primary tumors and lymph node metastases. In patients with ALDH1⁺ primary tumors and lymph node metastases (n = 7), immunohistochemical study of ALDH1 in lymph node metastases was carried out for a comparison with primary breast tumors.

Figure 7

Relapse‐free survival (RFS) curves of breast cancer patients according to aldehyde dehydrogenase (ALDH) 1 expression. (a) RFS rates were compared for patients with ALDH1⁻ tumors and those with ALDH1⁺ tumors. Patients with ALDH1⁺ tumors were categorized into those with ALDH1 (1+ and 2+) tumors and those with ALDH1 (3+) tumors, and their RFS rates were compared with those of ALDH1⁻ tumors (b) and (c), respectively.