The cancer stem cell hypothesis has fundamental implications for cancer biology in addition to clinical implications for cancer risk assessment, early detection, prognostication, and prevention. The development of cancer therapeutics based on tumor regression may have produced agents which kill differentiated tumor cells while sparing the small cancer stem cell population (Wicha et al., 2006
). The development of more effective cancer therapies may thus require targeting this important cancer stem cell population. The success of these new approaches hinges on the identification, isolation and characterization of cancer stem cells. Recently, the phenotype of the mouse mammary stem cells was identified by several groups (Shackelton et al., 2006
; Stingl et al., 2006
). These studies showed that an entire, functional mammary gland can be regenerated in vivo in several serial passages, starting from a single cell (Shackelton et al., 2006
). Also, considerable progress has been made recently towards identification of human mammary stem cells, although the phenotype of these cells has remained elusive (Clarke et al., 2006
, Villadsen et al. 2007
). Our study indicates that ALDH1 is a marker of stem/progenitor cells of the normal human breast and breast carcinomas. Utilizing in vitro assays we showed that ALDEFLUOR-positive cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential, capable of self-renewal. These cells also have the highest ability to grow in vivo, in a xenotransplantation animal model. In breast carcinomas, cells with high ALDH activity contain the tumorigenic cell fraction, able to self-renew and to recapitulate the heterogeneity of the parental tumor. With the caveat that xenotransplantation may change the properties of normal and cancer cells, this in vivo assay remains the gold standard for testing functional stem cell properties. The ALDEFLUOR-positive cell population has a small overlap with the previously described cancer stem cell, CD44+/CD24-/lin- phenotype. In the tumors we investigated, the overlap represented approximately 1% or less of the total cancer cell population. However, the cells bearing both phenotypes appeared to be highly enriched in tumorigenic capability, being able to generate tumors from as few as 20 cells.. It remains to be determined if these phenotypes are associated with stem cells in other breast cancers.
Identification of normal and malignant stem/progenitor cells by the same marker supports the concept that stem and progenitor cells are primary targets of transformation, and thus lends further support to the cancer stem cell hypothesis. In addition, the ability to identify stem/progenitor cells by this shared phenotypic trait, ALDH1 expression, permits analysis of cancer initiation and progression from the normal to the pre-malignant and then the malignant state. Unlike the previously described breast cancer stem cell phenotype, which requires the use of a combination of ten surface antigens (Al-Hajj et al., 2003
), testing for ALDH1 expression is a simple method for identifying normal and cancer stem cells, amenable to clinical applications. We showed in the present study that ALDH1 expression is a powerful prognostic factor for breast cancer and it has direct or inverse correlation with known histoclinical parameters, such as tumor grade, ER/PR status, ERBB2 overexpression and basal-like cytokeratins (CK 5/6 and CK14).
In the vast majority of breast tumors analyzed in this study the ALDH1 positive cells represented a relatively small population, consistent with the notion that cancer stem cells constitute a minority of the tumor population. Remarkably, only two tumors out of 481 analyzed, had a predominant ALDH1 positive population. These tumors had a very aggressive clinical evolution and may have been driven by a stem cell population locked in self-renewal, undergoing little or no differentiation.
We propose that ALDH1 expression in a subset of tumors may reflect transformation of ALDH1-positive stem or early progenitor cells in these tumors. By contrast, ALDH1-negative tumors may be generated by the transformation of ALDH1-negative progenitor cells. In the ALDH1-positive tumors, the cancer stem cell population may inherit properties of normal stem cells that confer aggressiveness: ability to self-renew, high proliferation potential, resistance to damaging agents and chemoresistance. This hypothesis is consistent with the studies of AML (Bonnet et al., 1999
). Alternatively, ALDH1-negative tumors may contain rare ALDH1-positive cells below the level of detection by immunostaining on TMAs. The detection of an ALDH1-positive population in TMA cores may be due to an increased self-renewal activity in these tumors. A recent study has shown that a gene expression signature associated with increased self-renewal of normal stem cells is a predictor of poor prognosis (Glinsky et al., 2005
; Lahad et al., 2005
). In agreement with our findings, previously described molecular signatures of breast cancer associated with a poor prognosis for breast cancer contain one or more ALDH isotypes (Alexe et al., 2006
). Recently, a combinatorial analysis of gene expression data was used to re-analyze the van’t Veer breast cancer gene expression data set (van’t Veer et al., 2002
). This analysis identified 17 genes associated with poor prognosis in breast cancer, two of which were ALDH isoforms. A recent study showed that granulocyte macrophage progenitor cells, transformed by the MLL-AF9 fusion protein, retained the global expression profile of their normal cells of origin and had only a subset of genes re-programmed. This set included 363 genes which were associated with self-renewal in normal hematopoietic stem cells including an ALDH isoform (Krivtsov et al., 2006
In conclusion, our study lends support to the cancer stem cell hypothesis, by showing that both normal and malignant mammary stem cells share a common functional marker, ALDH1. Identification of ALDH1 as a potential marker of normal and malignant human breast stem cells opens important new avenues of research in normal breast development and breast carcinogenesis. Furthermore, our study suggests that ALDH1 expression may be used to detect both normal and malignant mammary stem cells in situ, in fixed paraffin-embedded sections. The clinical utility and relevance of this assay was demonstrated by a strong association of ALDH1 expression with clinical outcome in two independent tumor sets, totaling 577 patients. Since ALDH is also expressed in hematopoietic and neuronal stem cells, this marker may prove useful for the detection and isolation of cancer stem cells in other malignancies, thus facilitating the application of cancer stem cell biology to clinical practice.