NGAL appears to protect MMP-9 from autodegradation, increasing its activity by binding and forming MMP-9/NGAL complexes. Tumor cells excrete elevated levels of NGAL resulting in an increase in local concentration of MMP-9, which can affect various aspects of tumor progression . NGAL is abundantly expressed in adipose tissue and liver and recent studies have correlated circulating NGAL levels with obesity and its metabolic complications [32, 33]. NGAL has also been suggested as a marker of acute kidney injury [6–9]. Therefore normal liver and renal function was clinically confirmed for all patients included in this study prior to their enrollment. Moreover, no patients with metabolic syndromes participated in the study. According to our findings, serum levels of MMP-9, NGAL, and MMP-9/NGAL were not associated with obesity since no correlation with patients' BMI was observed.
Immunohistochemical studies on breast tissue have associated MMP-9 expression with a higher rate of distant metastases [34–36]. The prevention of the metastatic process is the main aim of the clinicians. Soluble biomarkers could be useful tools in the prediction of patient outcome and management of the disease. Therefore, MMP-9 detection in sera could provide significant information of the tumor biological features. Somiari and coworkers have suggested that circulating MMP-2 and MMP-9 levels are associated with disease severity and may permit the classification of patients with breast disease [24, 37]. These findings were recently extended from Wu et al, who reported that serum MMP-9 levels were significantly elevated in patients with breast cancer compared to those with benign breast disease and healthy controls. Additionally, increased MMP-9 levels were associated with lymph node metastasis, higher tumor stage, lower relapse-free and overall survival . In agreement with these observations, our study reported an increase in MMP-9 expression in patients with ADH, DCIS and IDC (p < 0.003) compared to healthy controls. Our results also document a positive correlation between MMP-9 serum levels and the severity score of breast disease (p < 0.006).
Recent evidence suggests that NGAL expression is associated with cancer invasive progression. Gene expression profiling and analysis of human pancreatic adenocarcinomas by cDNA microarrays, quantitative real-time RT-PCR and immunohistochemistry demonstrated increased expression of NGAL in malignant pancreatic tissue compared to normal . Lim et al reported that tissue expression of NGAL in ovarian tumors changes with disease grade and this is also reflected in serum levels . More specifically, tissue NGAL expression was undetectable in normal ovaries, weak to moderate in benign tissues, while it displayed highest levels in borderline and low-grade tumors. The authors also reported similar findings for NGAL expression in serum, with levels being significantly higher in patients with benign and grade 1 tumors compared to healthy controls. The role of NGAL was also investigated in esophageal squamous cell carcinoma (ESCC) and it was reported that its tissue expression was significantly higher in ESCC than in normal mucosa, and was positively correlated with cell differentiation . Based on their findings that NGAL in human tissue and urine samples were consistently associated with invasive breast cancer, Yang et al suggested that NGAL may be a potential noninvasive biomarker of breast disease . Our findings further support the hypothesis that NGAL is associated with cancer disease severity. Serum NGAL levels were higher in patients with IDC (p < 0.008) while moderate in patients with DCIS, ADH and sclerosing adenosis compared to healthy controls. A significant positive correlation between NGAL expression and breast disease severity score was also observed (p < 0.02).
Regarding MMP-9/NGAL, current evidence suggests that urinary detection of the complex may represent a new biomarker for the prediction of cancer disease [25, 26]. NGAL overexpression in human breast cancer cell lines was accompanied by increased tumor growth, MMP-9 activity, angiogenesis and cell proliferation. Moreover, MMP-9/NGAL enzymatic activity was observed in the urine of breast cancer patients but not in healthy controls . Evaluation of MMP-9 and MMP-9/NGAL complex in urine of patients with brain tumors revealed significantly higher expression levels compared to controls, which was also confirmed in tumor tissue. After tumor resection, clearing of biomarkers was observed. The findings support an association between MMP-9 and MMP-9/NGAL urine levels with the presence of disease and response to therapy . In our study, we attempted to evaluate the expression of MMP-9/NGAL complex in serum of patients with breast disease and correlate it with disease severity. Despite observing higher levels of MMP-9/NGAL in IDC (p < 0.01), DCIS, ADH and sclerosing adenosis patients compared to healthy controls, a positive non-significant correlation between complex expression and disease severity score was found. However, future studies including a higher number of patients could elucidate the significance of serum MMP-9/NGAL expression in patients with breast disease.
The relation between breast benign diseases and invasive cancer has been a matter of discussion for many years, as these lesions are considered precursors in malignant transformation. Sclerosing adenosis is a benign proliferative lesion without atypia with a relative risk for malignant breast disease of 1.3-1.9, while ADH has a slightly higher risk (× 3.9-5.0) . Since sclerosing adenosis and ADH are believed to constitute distinct precursors of invasive breast carcinoma, we attempted to investigate potential differences in the tested parameters. Higher levels of NGAL and MMP-9/NGAL complex were observed for both groups of patients compared to healthy controls, whereas MMP-9 expression was particularly elevated in the sclerosing adenosis group. This observation seems to support the idea that this lesion is a distinct entity of benign disease. Whether serum levels of MMP-9, NGAL and their complex can reflect the relative risk of patients with benign diseases for developing breast cancer remains to be investigated in future studies.
Improved non-operative diagnostic techniques enable the detection of breast cancer at an earlier stage. At the same time, serum measurements represent a non-invasive, easily accessible method for the study of biomarkers as screening tools for risk assessment, diagnosis and prognosis of breast cancer. The present study was designed in an effort to reveal specific biomarkers associated with breast disease progression. Our data suggest that MMP-9 and NGAL are positively correlated with breast disease severity, with a potential clinical utility as early markers of breast disease status.