Abstract
BackgroundWorldwide more than one million women are annually diagnosed with breast cancer. A considerable fraction of these women receive systemic adjuvant therapy; however, some are cured by primary surgery and radiotherapy alone. Prognostic biomarkers guide stratification of patients into different risk groups and hence improve management of breast cancer patients. Plasma levels of Matrix Metalloproteinase-9 (MMP-9) and its natural inhibitor Tissue inhibitor of metalloproteinase-1 (TIMP-1) have previously been associated with poor patient outcome and resistance to certain forms of chemotherapy. To pursue additional prognostic information from MMP-9 and TIMP-1, the level of the MMP-9 and TIMP-1 complex (MMP-9:TIMP-1) was investigated in plasma from breast cancer patients.MethodsDetection of protein:protein complexes in plasma was performed using a commercially available ELISA kit and, for the first time, the highly sensitive in-solution proximity ligation assay (PLA). We screened plasma from 465 patients with primary breast cancer for prognostic value of the MMP-9:TIMP-1 complex. Both assays were validated and applied for quantification of MMP-9:TIMP-1 concentration. In this retrospective study, we analyzed the association between the concentration of the MMP-9:TIMP-1 complex and clinicopathological data and disease free survival (DFS) in univariate and multivariate survival analyses.ResultsFollowing successful validation both assays were applied for MMP-9:TIMP-1 measurements. Of the clinicopathological parameters, only menopausal status demonstrated significant association with the MMP-9:TIMP-1 complex; P = 0.03 and P = 0.028 for the ELISA and PLA measurements, respectively. We found no correlation between the MMP-9:TIMP-1 protein complex and DFS neither in univariate nor in multivariate survival analyses.ConclusionsDespite earlier reports linking MMP-9 and TIMP-1 with prognosis in breast cancer patients, we here demonstrate that plasma levels of the MMP-9:TIMP-1 protein complex hold no prognostic information in primary breast cancer as a stand-alone marker. We demonstrate that the highly sensitive in-solution PLA can be employed for measurements of protein:protein complexes in plasma.
Highlights
Worldwide more than one million women are annually diagnosed with breast cancer
Efficiency of the pre-amplification was demonstrated by analyzing a plasma dilution curve with and without pre-amplification, which increased the Crossing point (Cp)-value with 15 points (Additional file 1: Figure A)
This procedure was investigated in one breast cancer plasma sample with low and one with a high level of Tissue inhibitor of metalloproteinase-1 (TIMP-1), determined by Enzyme-linked immunosorbent assay (ELISA) in a previous study [15], and it was found that implementation of pre-amplification was especially important when analyzing samples with low levels of target
Summary
Worldwide more than one million women are annually diagnosed with breast cancer. A considerable fraction of these women receive systemic adjuvant therapy; some are cured by primary surgery and radiotherapy alone. Plasma levels of Matrix Metalloproteinase-9 (MMP-9) and its natural inhibitor Tissue inhibitor of metalloproteinase-1 (TIMP-1) have previously been associated with poor patient outcome and resistance to certain forms of chemotherapy. Introduction of additional and validated prognostic biomarkers could add information to current risk stratifications resulting in a more effective management of future breast cancer patients. Concentrations of both Matrix metalloproteinase-9 (MMP-9) and the naturally occurring Matrix metalloproteinase (MMP) inhibitor, Tissue inhibitor of metalloproteinases-1 (TIMP-1), have been investigated as tumor biomarkers in breast cancer [4,5,6,7,8]. The observed association between high TIMP-1 levels and poor prognosis may be explained by the other functions that have been disclosed for TIMP-1: influence on cell growth [18,19], angiogenesis [20,21,22], apoptosis independently of its MMP-inhibitory functions [23,24,25,26], and on epithelial-mesenchymal transition [27]
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