Introduction
Breast cancer is associated with a considerable amount of distress for women, as well as with a risk of mortality worldwide (1). It is the major cause of cancer-related mortality among Iraqi patients, accounting for approximately one-third of all cases of cancer registered in the country (2). Breast cancer is currently considered as a group of heterogeneous diseases that vary in their clinical behavior, response to therapy and outcome. Even with the progress being made in diagnosis and treatment that favors early management, metastasis is inevitable for certain patients, rendering breast cancer a major burden on public health (3,4).
Cancer stem cells (CSCs) are a special type of population of cancer cells that carry characteristics of stem cells; they may also initiate the development of breast cancer. CSCs give rise to daughter cells at any time during cell division that can occur following the self-renewal of these stem cells, maintaining the full capability to divide and differentiate from the ordinary tumor cells that represent the major element of the tumor (5).
The need to identify specific biomarkers for CSCs has been considered in previous studies. A number of proteins have been identified as promising markers, including nestin, CD133, CXCR-4, EpCAM, ABCB1, aldehyde dehydrogenase 1 (ALDH1), Oct-4, c-Met, ABCG2, CD24 and CD44 (5-7).
The present study evaluated the expression of the representative CSC markers, CD133 and ALDH1, using immunohistochemical staining and their distribution in invasive (ductal) carcinoma of the breast. The present study also assessed their association with clinical and pathological parameters of patients, such as the age, stage and tumor grade tumor, as well as hormonal receptor state [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)/neu].
Prominin-1 (also known as CD133) is one of the key members of pentaspan trans-membrane glycoproteins. The CD133 encoding gene in human beings is located on chromosome 4(8). Its expression is frequently found on multipotent primitive progenitor cells and immature hematopoietic stem cells. CD133 is an acknowledged marker for CSCs.
The tumorigenic role of CD133 expression can be identified when CD133-expressing cells exhibit the majority of features of stem cells, such as self-renewal ability, a high proliferation rate and resistance to cancer drugs. This marker may also participate in cell differentiation, proliferation and apoptosis (9,10).
CD133 is one of the markers that can be used in the, as well as in the prognosis of various neoplasms. In breast cancer, the increased expression of this marker is relatively related to a poor prognosis. CD133 is a key promising marker for the identification of CSCs in breast cancer subtypes, including the aggressive HER2-positive and triple-negative phenotypes (11).
ALDH1 belongs to a NADP-dependent enzyme family as a detoxifying enzyme that plays essential roles in the oxidation of extracellular and intracellular aldehydes to their corresponding carboxylic acids. It is essential in protecting cellular homeostasis by scavenging the reactive aldehydes that result from lipid peroxidation (12). A high expression level of ALDH1 is associated with resistance to cancer drugs by interfering with the cytotoxic effects of drugs used in cancer treatment (13).
Beyond their utility as diagnostic indicators, CD133 and ALDH1 function as active drivers of the aggressive phenotypes observed in invasive ductal carcinoma. CD133 (prominin-1) is not merely a surface protein; it functions as a critical scaffold that modulates intracellular signaling, specifically by activating the PI3K/Akt and Wnt/β-catenin pathways to promote self-renewal and epithelial-mesenchymal transition (9,12).
Simultaneously, ALDH1 plays a vital metabolic role by catalyzing the oxidation of retinol to retinoic acid, a potent regulator of gene expression that maintains cells in an undifferentiated, stem-like state. Furthermore, ALDH1 provides a ‘metabolic shield’ by detoxifying reactive oxygen species and metabolic byproducts of chemotherapy, such as aldehydes, thereby directly facilitating resistance to treatment and a rapid cellular turnover characteristic of high-stage, hormone-insensitive tumors (11,13).
Discussion
In the present study, the expression and distribution of the CSC markers, CD133 and ALDH1, in breast invasive ductal carcinoma were evaluated. Immunohistochemical analysis revealed that CD133 expression was localized to the membrane and cytoplasm of tumor cells. By contrast, this expression was absent in adjacent histologically normal breast tissue. The rate of expression progressively increased from normal to non-invasive to invasive carcinoma. These findings align with patterns previously observed in studies from Egypt and China (16,17).
In the present study, the analysis detected CD133 expression in 65.4% (34/52) of cases, consistent with the findings of a previous study in Egypt (68%) (16). Furthermore, the increased expression of CD133 exhibited a positive association with higher grade tumors (P=0.01) and an advanced disease stage (P=0.006). These associations align with previous research from Egypt (P=0.008 and P=0.002, respectively) by Ahmed and Mohammed (16).
The significant association between CD133 expression and an advanced histological grade (P=0.01) identified in the present study suggests that CD133-positive cells contribute to the loss of cellular differentiation. This supports the hypothesis that grade III tumors harbor a larger pool of CSCs, which drive aggressive biological behavior and rapid cellular turnover (18,19).
The enrichment of CD133 in advanced disease stages may be attributed to the survival advantages of CSCs. These cells exhibit resistance to cell-detachment-induced apoptosis, allowing them to survive lymphatic transport and establish secondary tumor deposits, which defines the clinical progression to stage III (19-21).
In the present study, significant associations were observed between CD133 expression and a negative ER status (P=0.031), a negative PR status (P=0.011). These findings align with previous reports from cohorts in Egypt and Korea (16,22). Conversely, no significant association was found between CD133 expression and patient age (P=0.119), a result consistent with prior studies in China (18).
A potential explanation for these findings is that ER/PR-negative tumors tend to be more undifferentiated, resembling normal breast tissue less than their hormone-receptor-positive counterparts, rendering them inherently more difficult to treat. This suggests that CD133-positive CSCs are more prevalent in triple-negative or hormone-insensitive subtypes, ultimately contributing to a poorer prognosis. Furthermore, evidence suggests that HER2 signaling actively drives the expansion of the CSC population. In these tumors, CD133 and HER2 likely function synergistically to fuel rapid cellular turnover and promote resistance to standard chemotherapy (22,23).
In the present study, ALDH1 expression was infrequent in normal breast tissues matched with cancer tissues. Furthermore, it was found that ALDH1 expression was in 67.3% of cases, in agreement with this finding, (68.8%) cases were reported in another Iraqi study on breast cancer (24). A significant association was also observed between ALDH1 expression and a high tumor stage (P=0.014); this finding aligns with previous studies in Korea (P=0.011) (22) and China (P=0.043) (23). By contrast, ALDH1 expression was not significantly associated with age (P=0.067) or histological tumor grade (P=0.57). These non-significant findings are supported by the findings of previous studies in China (P=1.000 and P=0.508) (25) and Africa (P=0.731 and P=0. 0.421) (26).
The findings of the present study indicate a biological decoupling between cancer cell stemness and cellular differentiation, suggesting they are regulated by independent genetic pathways. This divergence explains why a tumor can exhibit high invasiveness and an advanced clinical stage, driven by a robust stem cell population, while still maintaining a relatively organized histological structure or moderate grade (26).
The results presented herein demonstrate a significant association between ALDH1 expression and the ER (P=0.004) and PR (P=0.014) status. However, no significant association was found with HER2 expression (P=0.134). These findings align with the meta-analysis conducted by Liu et al (27), which similarly confirmed a robust association with ER (P<0.00001) and a non-significant association with HER2 (P=0.06).
The significant association between ALDH1 and the ER/PR status, which was in contrast to the lack of an association with HER2, may reflect the origin of these tumors from ALDH1-positive luminal progenitor cells. This suggests that ALDH1-driven stemness in the present study cohort was integrated into the hormone-signaling architecture of the tumor, rather than being driven by HER2-mediated growth factor pathways.
The present study had certain limitations, which should be mentioned. While the observed associations provide valuable insight, several limitations warrant consideration. First, the retrospective design and modest sample size may constrain the generalizability of these findings across more diverse patient populations. Nonetheless, these results establish an essential exploratory baseline. Second, the modest sample size (n=52) and the inclusion of only Iraqi patients may constrain the generalizability of these findings to broader, more diverse populations. Third, and most critically, the present study lacks patient outcome data, including survival analysis, recurrence rates and treatment response information.
However, the strong association between CSC markers and aggressive tumor characteristics suggests a potential link to treatment resistance, providing the necessary preliminary evidence to justify large-scale, prospective studies. The data presented herein highlight CD133 and ALDH1 as high-priority candidates for future research into predictive biomarkers and targeted therapeutic interventions.
In conclusion, the concomitant elevation of CD133 and ALDH1 expression is significantly associated with advanced clinical stages of breast cancer. As robust indicators of biological aggressiveness in invasive ductal carcinoma, these markers carry substantial prognostic value and may inform the selection of molecularly targeted therapies. Furthermore, the high prevalence of CD133 in aggressive phenotypes, particularly those characterized by hormone receptor (ER/PR) negativity and HER2 overexpression, underscores its role in driving therapeutic resistance and tumor recurrence. Consequently, characterizing these CSC populations may facilitate the development of more precise, evidence-based strategies to overcome chemoresistance.