MMP-11 expression in early luminal breast cancer: associations with clinical, MRI, pathological characteristics, and disease-free survival

In our investigation involving 228 women diagnosed with early-stage invasive ductal carcinoma of luminal types A and B, immunohistochemical analysis revealed that more than 50% of the tumors exhibited MMP-11 expression, predominantly within fibroblast-shaped cells present in the tumor stroma, known as cancer-associated fibroblasts (CAFs) [28].

A comprehensive review of clinical research on MMP-11 expression in human breast cancer, as summarized in Table 3, indicates a predominant reliance on immunohistochemistry for its detection. Comparatively, fewer studies have employed techniques such as northern blotting to assess RNA levels in tissue samples or in situ hybridization for detecting RNA on histological sections. Two studies [10, 29] performed both immunohistochemistry and in situ hybridization on a subset of cases, showing closely similar patterns of positivity. Exploiting data from publicly available databases, we observed a weak correlation between mRNA and protein levels for MMP-11. This disparity could be attributed to factors such as poor protein stability MMP-11 has a strong autoproteolytic activity [30] and is degraded by MMP-14 [14]. It is also important to note that while the detection of the protein itself is valuable, as MMP-11 is a proteolytic enzyme, the measurement of its enzymatic activity would provide a more direct assessment of its biological activity. However, currently, there is no robust system to make this kind of study.

The number of MMP-11 positive cases found in this study is consistent with previous studies considering similar patient cohorts [31, 32]. The majority of past studies have identified MMP-11 predominantly within the tumor stroma, specifically in elongated, fibroblast-like cells located either intermingled with cancer cells in the center of the tumor or in the periphery in the invasive front [10, 12, 24, 31, 33, 34].In these studies MMP-11 staining was always intracellular, this can be explained by the rapid degradation of MMP11 in the extracellular space by an auto-degradation process or by other MMPs such as MMP14 [14, 30]. Our findings highlight the presence of MMP-11 in the tumor stroma, where it likely exerts its functional effects. Cancer-Associated Fibroblasts (CAFs) are increasingly recognized as key modulators of the tumor microenvironment of several types of solid tumor, including breast cancer. They exhibit vast molecular and genetic variations and are recruited from distinct sources. Some are native to the breast stroma, originating from resident tissue fibroblast or de-differentiated adipocytes, others may arise from the perivascular space during tumor evolution, or directly from tumor cells through epithelial-to-mesenchymal transition [35]. Distinct CAF subsets have been correlated with specific breast cancer molecular subtypes and prognostic value [36]. Although the classification of CAF subtypes has not reach a consensus yet among various studies, there is a general agreement on two main subtypes: myocontractile CAFs (myCAFs) that secrete extracellular matrix, and inflammatory CAFs (iCAFs), noted for their immunomodulating functions [27].

Intriguingly, several studies also reported MMP-11 expression within tumor cells themselves [10, 12, 32, 34, 37]. This expression pattern has been associated with certain breast cancer subtypes, like invasive lobular carcinoma [37] or metaplastic carcinoma of the breast [10], both tumor types characterized by an epithelial-to-mesenchymal transition (EMT) phenotype. In other studies, MMP-11 was found expressed in tumor cells of traditional invasive ductal carcinoma [12, 32, 34], though the epithelial expression was much less constantly associated with prognosis than the stromal expression. Similar findings have also emerged for other cancer types, including MMP-11 expression in spindle-shaped tumor cells of oral cavity cancers [38], in prostate and pancreatic tumor cells [39, 40]. In our study, we observed the absence of epithelial MMP-11 expression, which could be attributed to the specific composition of our cohort, notably the exclusion of lobular invasive carcinoma and rare subtypes. We also considered the potential cross-reactivity of the MMP-11 antibody with other MMPs, such as MMP2 (gelatinase A), MMP9 (gelatinase B), and MMP14 (membrane-type 1 MMP), which are commonly associated with tumor cells. This cross-reactivity may result in false-positive staining in epithelial cells. Analysis from a comprehensive single-cell database reinforced our findings, indicating that stromal cells, particularly CAFs and perivascular cells, are the predominant sources of MMP-11 expression.

Our research stands out as the only study to specifically concentrate on hormone receptor (HR)-positive breast cancers, the most common subtype. Previous reports either did not explicitly mention the proportion of HR-positive cases [9, 12, 24, 33, 34] or found them to constitute between 48% and 68% of their study populations [12, 32]. Interestingly, our analysis revealed a direct correlation between a high MMP-11 immunohistochemical score and an elevated tumor nuclear grade—a connection also supported by earlier studies that included a whole range of clinical and molecular breast cancer subtypes. Furthermore, parallels were drawn between overexpression of MMP-11 and increased markers of proliferation, such as Ki67 or TopoIIα [34]. Rapidly proliferating tumors necessitate enhanced metabolic support from their adjacent tissues. While MMPs are traditionally understood to remodel the extracellular matrix, the elevated expression of MMP-11 could also play a pivotal metabolic role. This could be mediated through a paracrine action on neighboring adipocytes, as indicated by previous studies [41‐43]. Our study reinforces the idea that MMP-11 plays a pro-tumoral role in hormone-receptor positive breast cancers, the predominant subtype of breast cancers.

Our analysis identified a novel association of MMP-11 expression with specific peritumoral texture-related features on MRI, an aspect not explored in earlier studies. Breast MRI data analysis links peritumoral stroma characteristics with MMP-11 expression levels, suggesting a potential impact of MMP11 on peritumoral fat. The function of MMP-11 the adipose tissue and on adipocyte dedifferentiation has been documented experimentally in cells and animal studies [44]. This finding could open new avenues for non-invasive assessment of tumor biology using advanced imaging techniques, in particular MRI [45]. It is already known that MRI-detected peritumoral edema is associated with lymphovascular invasion, tumor necrosis and stromal fibrosis [46] and quantitative assessment of the peritumoral fat has been linked to cancer subtype [47].

The Urokinase plasminogen activator (uPA) plays a pivotal role in reshaping the extracellular matrix by converting plasminogen to plasmin, which subsequently activates various MMPs. While no direct molecular link between uPA and MMP-11 has been identified, their combined expression has been associated with an unfavorable prognosis [33]. The statistical association between the expression of these two proteases reinforces the idea that a matrix-associated protease activation cascade occurs in certain breast cancer types at an early stage.

Importantly, in terms of disease-free survival, our study noted that high MMP-11 expression, particularly when combined with a high Ki67 index, suggests an association with reduced DFS, even in the presence of a favorable overall prognosis, as demonstrated by the limited number of oncological events. This complements previous research using immunohistochemistry, such as [24] and [34], or RNA studies: for example, MMP-11 has been identified as part of a distinguishing genomic signature of “metastasis-associated fibroblasts” [48], a genomic signature related to breast cancer progression [49] and a genomic signature associated with the risk of recurrence of tamoxifen-treated, node-negative breast cancer [6]. Similarly, the study by Eiró et al. [50] further corroborates the importance of MMPs and TIMPs expressions within the fibroblastic compartment in stratifying prognostically significant microenvironment clusters. Ki67 was also identified as a significant prognostic factor in our study, where a 15% cutoff proved most effective for discrimination, aligning with findings from earlier research [51]. Notably, the combined analysis of Ki67 and MMP-11 in our cohort markedly improved prognostic accuracy. This was particularly evident as only one out of 125 patients with low expression levels of both Ki67 and MMP-11 experienced recurrence, compared to 11 out of 103 patients showing overexpression of either protein. These observations were recorded over a median follow-up period of 6.3 years.

Before implementing MMP-11 combined with Ki67 detection in the routine practice, external validation is needed in a prospective cohort. An online analysis of publicly accessible survival data from www.​kmplot.​com, encompassing 1496 hormone receptor-positive breast cancer patients selected based on endocrine therapy, reveals that higher RNA expressions of both Ki67 and MMP-11 are independently linked to increased recurrence risk in multivariate analysis. Specifically, higher Ki67 expression corresponds to a relative risk (RR) of 1.39 [95% CI: 1.11–1.73, p = 0.0042], while elevated MMP-11 expression is associated with a RR of 1.27 [95% CI: 1.01–1.58, p = 0.038]. Although these findings are based on RNA expression rather than protein levels, they still underscore the prognostic significance if MMP-11 in luminal breast cancer, suggesting of its potential as a biomarker, even when different methodologies are employed.

The management of breast cancer patient is constantly evolving: for HER2-enriched or high-grade triple-negative breast cancers, the administration of neoadjuvant or adjuvant systemic therapies is a common approach to reduce recurrence rates. In contrast, luminal tumors of similar size generally have a more favorable prognosis and some researchers suggest reconsidering the necessity of adjuvant chemotherapy [52] or even adjuvant endocrine therapy [53] in low-risk patients. Tumor size continues to be a critical factor, which is why our study predominantly focused on T1 tumors. A significant portion of the tumors included in our analysis fell within the 1 to 2 cm size range, mirroring the populations observed in other research dedicated to de-escalation strategies [52]. This stratification approach agrees with the broader goal of personalized medicine in oncology, where treatment decisions are guided by specific biomarkers.

Given its relative simplicity and low cost, immunohistochemical evaluation of MMP-11 expression in tumor tissues offers a complementary prognostication tool, allowing for a more nuanced risk assessment, tailored follow-up of these early, hormone receptors positive, breast tumors.

Our study has some limitations. Despite being one of the more extensive clinical studies on this topic, the limited number of oncological events may hamper its power to highlight significant statistical differences. Additionally, the immunohistochemical assessment’s semi-quantitative nature, though straightforward, can introduce potential inter-reader variability and might be affected by technical inconsistencies. This study did not primarily focus on tumor heterogeneity, but it is important to recognize that a low MMP-11 immunohistochemical score can be associated with localized MMP-11 expression, which might indicate local progression, through MMP-11 function on substrates like IGFBP1 and Collagen VI [13, 54]. However, it is worth noting that currently, we lack a straightforward method to concurrently assess the presence of MMP11 and its substrates in tumors. Eventually, given the lack of specific fibroblast marker, we relied on cell morphology to identify CAFs, which is a common issue when studying fibroblasts [27].