Plasma Matrilysins MMP-7 and MMP-26 Concentrations as Diagnostic Biomarkers for Breast Cancer Patients

Background: Metalloproteinases (MMPs) are a group of proteolytic enzymes involved in the maintenance of a proper structure of extracellular matrix (ECM). Matrilysins (MMP-7 and MMP-26) are the one of the group of MMPs that could represent potential breast cancer (BC) markers. The aim of the study was to evaluate plasma levels of MMP-7, MMP-26 and CA 15-3 individually and in combination and assess the a diagnostic utility of studied matrilysins in BC patients. Methods: The study group consisted of 120 patients with BC, the control group consisted of 40 patients with benign breast cancer and 40 healthy women. Concentrations of MMP-7 and MMP-26 were determined by Enzyme-Linked Immunosorbent Assay, CA 15-3 by Chemiluminescent Microparticle Immunoassay. Results: The plasma levels of MMP-7 were signicantly higher in the entire BC group than in the control group. Concentrations of MMP-26 and CA 15-3 were the highest in the III and IV stage of disease. The highest diagnostic sensitivity was observed in the III and IV stage of cancer for set of all tested markers (92.5%). The highest diagnostic specicity was noted for all tested parameters in all studied BC group (95.0%). The area under the receiver operating characteristic (ROC) curve (AUC) set of markers (MMP-7+MMP-26+CA 15-3) was the largest (0.9138) in III and IV stage. Also individual marker analysis showed that MMP-7 had the highest AUC (0.8894) in advanced stages of disease. Conclusions: Data suggested that MMP-7 can be considered as additional marker improving diagnostic utility of CA 15-3 in early stages of BC patients. Therefore, combined analysis of MMP-7 and MMP-26 with CA 15-3 might be useful in detection of disease progression. Future investigation is needed to evaluate whether matrilysins might be a potential markers improving diagnosis of BC.


Background
Breast cancer (BC) is the most often diagnosed cancer in female around the world. It mainly originates from the lactiferous ducts as a result of uncontrolled proliferation in epithelial cells (1). Studies of pathological processes associated with tumour growth and the occurrence of lymph node metastases and distant metastases revealed matrix metalloproteinases (MMPs) as pivotal proteins involved in shaping the tumour microenvironment and thus, cancer progression and metastases (2,3). MMPs are the family of a proteolytic enzymes responsible for the remodelling of the extracellular matrix (ECM). Most of MMPs consist of a propeptide, a catalytic metalloproteinase domain, a linker peptide of variable lengths and a hemopexin (Hpx) domain. MMPs include matrilysins: MMP-7 and MMP-26, which do not have the linker peptide and the Hpx domain (4). MMP-7 may affect the structure of casein, laminin, bronectin, collagen III/IV/V/IX/X/XI, type I/II/IV/ V gelatins, elastin and proteoglycans inducing their degradation (5). MMP-7 also regulates several biochemical processes such as activation, degradation, and shedding of non-ECM proteins. Heparin-binding epidermal growth factor precursor (proHB-EGF), membrane-bound Fas ligand (FasL), tumour necrosis factor (TNF) alpha precursor, E-cadherin are cleaved by MMP-7 into mature HB-EGF, soluble FasL, TNF-alpha and E-cadherin which promote cellular proliferation and invasion (6). MMP-7 is secreted speci cally by epithelial cells and the tumour cells themselves, which is opposite to the other MMPs, which are mostly produced by macrophages, endothelial cells or broblasts (5,6).
MMP-26 cleaves of not only ECM components e.g. vitronectin, brinogen, type IV collagen and gelatins, but also non-ECM proteins such as insulin-like growth factor-binding protein-1 (IGFBP-1) and α-1 protease inhibitor (7,8). Expression of MMP-26 in healthy tissues is reduced, whereas its expression increases in cancerous tissue of epithelial origin (9,10). Data performed by Marchenko et al. have shown increased MMP-26 gene expression in various tumour cell lines including MCF-7 breast carcinoma cells. In addition, they speculated that MMP-26 is probably involved in destruction of necrotic tissue of oxygen de ciency tumours and may participate in neovascularization and angiogenesis (10).
Available literature data most often described increased matrilysins expression in breast cancer cells, but there are few reports connected with their plasma concentration in breast cancer patients. Therefore, the aim of the current study was was to determine the plasma levels of MMP-7 and MMP-26 in comparison to the commonly accepted tumour marker (CA 15 − 3) at various stages of BC.

Methods
Enrolled patients Table 1 shows the study and control groups. The study group comprised 120 patients with BC referred to the Department of Oncology, Medical University of Bialystok, Poland, between 2015 and 2018.
Classi cation and assessment of the stage of the tumour were established in accordance with the International Union against Cancer Tumour-Node-Metastasis (UICC-TNM) classi cation. Histopathology of breast cancer was assessed in all cases by biopsy of mammary tumour tissue before or after surgery (all patients with ductal adenocarcinoma). Written consent, including participants' own statements about their medical history (i.e. data related to reproductive history, personal or family history of cancer, general health problems: hospitalization or surgery and the use of drugs) and lifestyle habits, including smoking, were obtained. None of the patients received chemotherapy or radiation therapy before taking blood samples. Initial assessment procedures had included physical examination and blood tests, mammography, breast ultrasound, breast core biopsy and chest x-ray. In addition, radioisotope bone scans, bone marrow aspiration and examination, and brain and chest tomography scans were performed as needed. The control group consisted of 80 cases divided into 2 groups: 40 patients with benign breast lesions and 40 healthy patients. All patients in the control group underwent mammary gland examination by a gynaecologist and breast ultrasound. The benign mammary lesions were con rmed by histopathological examination. In addition, women with in ammation and associated diseases such as circulatory disorders were excluded from the study.
The study was approved by the local Ethics Committee (R-I-002/51/2015) and all patients agreed to participate in the study. Comparison of the diagnostic power of all studied markers was assessed using the areas under the receiver operating characteristic (ROC) curve (AUC) which were performed using the GraphRoc program for Windows (Windows, Royal, AR, USA). Healthy patients and benign breast tumour groups represented the control group in analyses of diagnostic performance (SE, SP) and ROC curves.  The concentrations of MMP-7, MMP-26 and CA 15 − 3 in BC group were signi cantly higher than in total control group (benign breast tumour and healthy patients) (p < 0.001). The median levels of all tested parameters in stage III and IV were increased in comparison to control group (p < 0.001). Moreover, the concentrations of MMP-7 in stage II were statistically higher than in control group (p = 0.013).  The relationship between the diagnostic SE and SP is illustrated by the ROC curve in Table 4. The AUC indicates the possible clinical usefulness of a tumour marker and therefore, its diagnostic power. In the total group of BC AUCs for all parameters were signi cantly higher in comparison to AUC = 0.5 (p < 0.001).

Discussion
MMPs have proven role in BC progression. They participate in the modulation of the immune system, angiogenesis and development of the tumour microenvironment, leading to the progression of cancer. Their ability to disintegrate ECM components is considered as key factor leading to disease progression (11). Among MMPs, the most extensive studies to date have been conducted on MMP-2 and MMP-9, which are considered to have a signi cant impact on the development of BC (12)(13)(14). Nevertheless, is essential to search the functions of other MMPs that may further explain their role in BC progression. In this paper we focused our attention on serum levels of MMP-7 and MMP-26 in BC patients. In addition, we compared the tested enzymes with the commonly marked protein The data presented here revealed that BC patients demonstrate a signi cantly higher concentrations of plasma levels MMP-7, MMP-26 and CA 15 − 3 than healthy patients. Moreover, concentration of MMP-7 was increased in patients with benign breast tumour in comparison to healthy subjects. We hypothesize that serum concentrations of studied parameters may be predictive factors when distinguishing between healthy and BC patients or even benign breast changes. Consequently, patients with III and IV stage of disease had a signi cantly higher MMP-7, MMP-26 and CA 15 − 3 levels when compared to I stage of BC and control group. Literature data provided no reports on plasma concentrations of matrilysins in patients with BC. Clinical studies suggest that circulating MMPs may indicate early signs of BC (15,16). Nevertheless, clinical-control cohort studies regarding the relationship between the concentration of MMPs include MMP-7 in plasma and the subsequent risk of postmenopausal breast cancer showed no dependence. No difference between concentration in the study group and control was observed. There was also no difference between the concentration of MMPs and the occurrence of mammary cancer (16).
Consistent with data cited above is study performed by Aroner et al. (17). They also performed 10 years follow-up study associating plasma MMPs, e.g. MMP-7 witch BC risk. Additionally, no signi cant association of these MMPs with BC subtypes was found, although a positive association with node metastases for MMP7 was suggested. This indicates that MMP-7 is not suitable to be a marker for detection of early stages of BC; however, it seems to be an appropriate marker for diagnostic and therapy monitoring in advanced stages of BC (17).
Katunina et al. performed analysis of MMPs (including MMP-7) in tumour tissue, adjacent histologically intact tissue and serum of patients with BC (18). Enzyme immunoassay test showed higher MMP-7 levels in cancer than in healthy tissue. There was no correlation between concentration MMP-7 in tissue and serum. Serum analysis did not show a signi cant increase in MMP-7 concentration in BC subjects compared to the control group (18). Their results are oppose with our observations. Nevertheless, it is worth noting that the researchers conducted analysis in serum not in plasma as we did. In addition, they had a small study group − 45 women with breast cancer and 8 patients in the control group. Therefore, due to study limitations, the results they received may explain no signi cant relationships between the tested groups.
It is known that MMP-26 is involved in the development of estrogen-dependent cancers, including breast cancer (19,20). and MMP-26 in plasma and serum of the umbilical cord blood (21). However, work of Galewska is not associated with breast cancer, so we are unable to compare our ndings regarding MMP-26 or both enzymes since no reports on the subject are available.
Sensitivity, speci city and area under ROC curve characterise the diagnostic usefulness for tumour markers. Considering our data higher values of SE for all tested parameters were observed. The SP for individual matrilysin and CA 15 − 3 was the same in all studied group (95%). Similar results were received for MMP-7 by Będkowska et al. who analysed MMP-7 concentration in epithelial ovarian cancers (22). They observed higher concentrations of MMP-7 compared to the healthy group. Moreover they noted higher SE values associated with tumour progression. The SP was the same in all disease stages (95%). Considering AUC values, they showed signi cantly higher AUCs when compared to AUC = 0.5 in all studied ovarian cancer group (22). In our work the AUCs of all compared markers were signi cantly higher compared to AUC = 0.5 in II, III and IV stage of cancer. In I stage of advancement only single analysis of MMP-7 or in conjunction with MMP-26 and CA 15 − 3 demonstrated diagnostic usefulness. Work of Leelawat et al. described diagnostic utility of MMP-7 in cholangiocarcinoma (23). They noted increased concentration of MMP-7 in serum of cholangiocarcinoma patients and SE and SP values were higher for matrilysin than for commonly used marker in diagnosis of cancers of digestive tract CA 19 − 9. Interestingly, analysis of AUC showed that MMP-7 was more accurate than CA 19 − 9 in diagnosis of cholangiocarcinoma (23). Vocka et al. studied accuracy of MMP-7 in diagnosing patients with metastatic colorectal cancer and compared serum levels of MMP-7 with CEA and CA 19 − 9 (24). Concentration of MMP-7 was signi cantly higher in patients with colorectal cancer compared to healthy controls. MMP-7 had very similar SE and SP as CEA, but had higher SE than CA 19 − 9. Serum level of MMP-7 correlated with worse disease outcome and had prognostic value (24). All results presented above described different types of cancer than BC, but showed similar trend e.g.

Consent for publication
Not applicable.

Availability of data
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.