In the present study, we found that patients with elevated lipocalin-2 or MMP-9 levels at diagnosis had poorer DFS than patients with low lipocalin-2 or MMP-9 levels. Women with a high level of lipocalin-2 (> 59.03 ng/ml) and MMP-9 (> 34.42 ng/ml) had significantly increased risk for recurrence or death than women with a low level of each protein. In addition, patients with both markers in high levels showed significantly poor DFS, especially in the women with low BMI or lymph-node negative breast cancer.
Although Noh et al. suggested the lymphovascular invasion, poorer histologic grade and higher nuclear grade are associated with poor DFS in Korean breast cancer cases in recently published paper , histologic grade and nuclear grade were not independently associated with DFS in our subjects. There could be several reasons for this varied survival profile such as relatively shorter follow-up period of our study, different baseline characteristics of study population and definition of outcome of both studies, and uncontrolled bias from method of tumor assessment.
Several studies investigated the serum level of lipocalin-2 and MMP-9 not only in patients with diseases but also in healthy controls. The previous observational studies have shown that the mean or median baseline value of lipoclalin-2 is highly contradictory dependent on the study design and assay method [21, 22]. The normal range of lipocalin-2 measured through our study (42.7-187.1 ng/ml) was comparable to the range suggested by Porta et al. (47–177 ng/ml ) [23, 24]. However, Choi et al. suggested the normal range as 43.8 ± 27.8 ng/ml which is much lower than our data (93.0 ± 29.9 ng/ml) .
Similarly the previous reports on the serum level of MMP-9 in healthy controls are highly contradictory [16, 26]. Although with a small sample size, Provatopoulou et al. demonstrated a marked increase in serum concentration of lipocalin-2 and MMP-9 for women with invasive ductal carcinoma compared with the healthy controls. This inconsistency across the studies might be caused mainly due to the method of sample collection (timing, storage, and preparation etc.), detection method and uncontrolled potential confounding factors related to patients’ characteristics. Furthermore, several factors related to the biochemical characteristics of each marker could have disturbed the concentrations of serum proteins such as the coagulation and fibrinolysis factor and leukocyte degraulation . In addition, given the sample size of our study, the standard deviation is too large, especially for both markers measured in cases, to obtain enough power to compare the concentrations between cases and controls. Thus our results should be interpreted with cautions and need to be confirmed in larger sample size. For this reason, we evaluated the variables based on the distribution of cases samples rather than to define the normal range of lipocalin-2.
In a previous study using tissue microarray containing samples from 207 breast cancer patients, elevated expression of lipocalin-2 correlates with some indicators of a phenotype severity including a low ER/PR expression, a low grade of differentiation, the presence of lymph-node metastases and a high Ki-67 proliferation index, and overexpressed lipocalin-2 is associated with poor DFS consistent with our results using serum [17, 28, 29].
The relationship between high levels of lipocalin-2 being detected in clinical samples of different tumor types, especially in epithelial origin tumors, such as esophageal , gastric , ovarian , pancreatic cancers  with clinical outcomes are being published. However, the precise source of serum lipocalin-2 has remained controversial. It is plausible that the overexpressed lipocalin-2 in tumor tissue released into the circulation may contribute to the elevated systematic level of lipocalin-2 supported by the direct correlation between elevated serum levels of lipocalin-2 and strong immunostaining grade in gastric tumor cells . In addition, as an acute-phase protein, lipocalin-2 may be released into the plasma from activated neutrophils, macrophages, and other immune cells in various inflammatory conditions [35, 36].
Recent findings suggest possible mechanisms underlying lipocalin-2 function in tumorigenesis, such as promoting the epithelial-mesenchymal transition (EMT) , regulating the iron homeostasis in cancer cells, affecting on steroid-dependent tumor growth and modulating MMP-9 activity . When lipocalin-2 is complexed with MMP-9, there is enhancement of the active MMP-9 pool . The cooperative role between lipocalin-2 and MMP-9 in breast cancer progression prompted us to investigate the concomitant presence of MMP-9 and we found the positive correlation between lipocalin-2 and MMP-9 in serum from breast cancer patients. Although several reports suggest that elevated serum levels of MMP-9 occur in serum and plasma of breast cancer patients [37–42], correlation with current clinical parameters and association with clinical outcome are still controversial mainly due to different types of samples being assayed and sampling procedures .
Furthermore, we observed the prognostic value is further intensified in certain subgroups. We found stronger and significant association in patients with a BMI less than 25 kg/m2. When we examined this association with a median value of BMI, the similar pattern was also observed although P for interaction was marginally significant. This finding may be partly explained by the fact that, among patients with a BMI less than 25 kg/m2, there was significant difference in lipocalin-2 levels between patients alive without recurrence and patients who had recurrence (P < 0.05). Given that obesity at diagnosis of breast cancer has been associated with an increased risk of recurrence and death [43, 44], it is possible that the impact of lipocalin-2 level and/or combined score on DFS could only be demonstrated in the lower BMI group, with these markers being independent prognostic factors of breast cancer.
In lymph-node negative breast cancer patients, the high score group was associated with a 5.36 fold increased risk compared with low score group. However, because of wide 95% CIs in these categorical analyses, these results should be interpreted with cautions. Yet the 95% CIs became narrower when examining analyses between ordinal value of risk group and DFS (HR, 2.21; 95% CI 1.45-3.35; P < 0.001). In our study, neither lipocalin-2 nor MMP-9 level correlated with lymph-node status. Assuming the cooperative role of lipocalin-2 and MMP-9 in tumor progression, poor prognosis associated with high levels of both markers could be attributed to the enhanced activity of MMP-9 by lipocalin-2 and subsequent molecular path related with tumoral invasiveness and diffusion in lymph-node negative status. To date, axillary lymph-node status remains the most powerful predictor for recurrence and survival of primary breast cancer. Nevertheless, approximately one-third of women with node negative breast cancer develop distant metastases 10 years after local therapy [45–47]. Thus, newer prognostic factors are urgently required to identify meaningful high risk subgroups within lymph-node negative breast cancer patients who may benefit from adjuvant therapy regimens. Although the hazard ratios of around 2 obtained from the combined risk score based on the overall patients are not big enough to impact on clinical practice, our result suggests that the combined score of both markers might have potential predictive value for metastasis and recurrence of patients with lymph-node negative breast cancer. However, these results are speculative and need to be confirmed in independent and large number of subjects to evaluate the external validity.
Our study has several limitations. We collected and measured both markers in a single manner, thus could not completely characterize the profiles of both markers accompanied by treatment response and subsequent survival. In addition, due to the relatively short term follow-up time, we could not assess the relationship between both markers and long term survival related to mortality of breast cancer. Thus these results must be interpreted cautiously and need to be confirmed in large prospective trials with serial measurements. In addition, it is worthwhile to investigate if surgical treatment of tumor results in subsequent clearing of both markers in serum assuming that the second primary cancer or recurrent breast cancer is causative for the elevated lipocalin-2 and MMP-9.