To the best of our knowledge, this is the first study reporting the relationship between ALDH1A1 nuclear expression and its clinicopathological features, as well as its prognostic impact in BC. The main findings of the present study were; (1) in those tumors with ALDH1A1 positive expression, 24% had nuclear staining, (2) the presence of obesity and the prevalence of smaller tumors were higher in patients with nuclear ALDH1A1 expression, and (3) patients with nuclear ALDH1A1 carcinomas appears to have better DFS and OS, although this was not statistically significant.
The presence of a cell population that exhibits characteristics of CSC has been described in various tumors (14–16), as well as in BC (17). These initiating cells possess the ability of self-renewal, differentiation and metastasis (18). The presence of these cells after standard therapies would explain the relapses in some BC patients. In 2005 Ginestier et al. determined the existence of a group of cells with increased ALDH1 activity in normal and cancer human mammary epithelial cells (4). In this study of breast carcinomas, ALDH1 positive cells exhibited tumorigenic characteristics such as the capacity of self-renewal and ability to generate tumors that resembled the heterogeneity of the original carcinoma, similar to those observed in CSC. These investigators also demonstrated the first the association of ALDH1 cytoplasmatic expression with clinical outcomes in BC.
Nevertheless, although cytoplasmatic ALDH1 expression has been well documented and proposed as a CSC marker, nuclear ALDH1A1 expression has been poorly reported. Outside the oncology field, the nuclear expression of ALDH has been identified in mammalian animal models in the keratinocytes of the cornea and in the retina. In the cornea, the presence of nuclear ALDH1 and ALDH3 seem to have a structural role in the development of its transparency during the postnatal period. It is believed to play a protective role through catalytic and non-catalytic mechanisms against oxidative stress generated by exposure to ultraviolet rays (19,20). Based on evidence regarding the inverse relationship between nuclear ALDH expression and rate of corneal proliferation, the regulatory role and maintenance of homeostasis in the corneal epithelium through the modulation of cell proliferation, and activation of differentiation programs, is attributed to this enzyme. This has been evident in animal models, to which, at 9 days in the postnatal period, the expression of nuclear ALDH3 is negative. At 14 days, during the same period, when the eye is opened and the proliferation of the corneal epithelium is reduced and reaches its normal thickness; ALDH3 is overexpressed (21).
In the oncology field, the inverse association of nuclear ALDH1A1 and rate proliferation has also been described in the adenoma- primary colorectal carcinoma- liver metastasis progression sequence as studied by Wang et al (13). In this study, ALDH1A1 nuclear expression was higher in low grade adenomas with low depth of infiltration, or negative lymph nodes. Patients with positive nuclear ALDH1A1 liver metastasis had better prognosis. Moreover, positive nuclear staining was consistent in primary lesions and their corresponding liver metastasis. However, Kahlert et al. identified positive nuclear staining of ALDH1A1 in 21 primary tumors in a colon cancer cohort (12). In this study, nuclear ALDH1A1 was associated with detriment in DFS and OS. Nevertheless, the authors did not consider the nuclear expression of ALDH1A1 as a prognostic biomarker useful in clinical practice due to the small number of patients showing this staining. In addition, this study also included patients with metastatic disease.
The hypothesis proposed by López et al in lung cancer that nuclear ALDHA1A1 expression plays a regulatory function in the cell cycle (11), as observed in the cornea, might explain in part its association with a higher response rates and a better prognosis. It could be that not only the diverse isoforms of ALDH perform different functions, but also the expression in different compartments of the same cells as well. Although more studies are needed to elucidate the exact function of the nuclear expression of ALDH1A1, taking into account the results of this study, this marker might have a predictive and prognostic role in BC.
In our cohort, a total of 18 (24%) BC cases had positive nuclear expression of ALDH1A1. When comparing positive nuclear staining to both cytoplasmatic staining and negative carcinomas, there were no significant differences in most of the variables included in this analysis. However, the presence of obesity was more frequent in patients with positive nuclear ALDH1A1 expression. Obesity has been associated with a proinflammatory microenvironment which can lead to the genesis of breast neoplasms. In preclinical models, the presence of fatty acid binding proteins has been related to induced ALDH1 enzyme activity in breast cancer stem cells (22). However, the relationship between obesity and ALDH1A1 nuclear expression had not been reported previously. More studies are needed to elucidate the role of this expression in BC patient with obesity. In addition, carcinomas with ALDH1A1 nuclear staining were smaller than those tumors that did not exhibit ALDH1A1 expression. As mentioned previously, the expression of nuclear ALDH1A1 in the cornea seems to be inverse to cell proliferation, so the loss of its expression could be expected in larger tumors that usually show increased cell rate proliferation. However, the small number of patients included in this analysis should be taken into account before drawing any conclusions.
According to the evaluation of the tumor after neoadjuvant chemotherapy, complete pathological response rate was superior in the positive versus the negative nuclear staining cases (66.7% vs 43.9%). However, this difference was not statically significant. Alternatively, in regards to the clinical outcomes, patients with positive nuclear ALDH1A1 staining had better DFS and OS, although this did not reach statistical significance (p log-rank test for DFS = 0.0519), probably due to the scarce sample size. This is in line with the data reported by López et al. who reported a median OS of 73 months in early stage lung patients with positive nuclear ALDH1A1 staining (11). Similarly, Wang et al. related nuclear ALDH1A1 expression with favorable prognosis in colorectal cancer (13). The reason for this association is not clearly understood. ALDH1A1 mainly catalyzes retinaldehyde to retinoic acid, which subsequently binds and activates the retinoic acid or the retinoid X receptors in the nucleus of the cell, thus promoting target gene expression. A hypothesis could be that ALDH1A1 nuclear expression downstreams genes of the retinoic acid pathway that are involved in the differentiation and proliferation of tumor cells, and may explain in part the trend to better prognosis in BC patients with positive nuclear ALDH1A1 staining observed in this study.
This study has several limitations, mainly due to its retrospective nature and its sample size. Regarding clinical implications, we could not firmly conclude that ALDH1A1 expression is associated with a better prognosis in BC patients. However, survival trend differences were consistently observed between both groups, and there were no differences in therapy strategies. We postulate that this hypothesis should be explored in further investigations with a higher sample size, as it could be a potential new biomarker for selected patients with BC. Otherwise, the physiopathological role of nuclear ALDH1A1, is not well understood or reported in the literature, and no conclusions should be made in this respect. Further studies could provide us a better knowledge of its function and its prognosis implications.