Abstract
Several studies have demonstrated an association between high tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and a poor prognosis of primary breast cancer patients. In the present study we investigated whether measurements of the uncomplexed fraction of TIMP-1 added prognostic information to that already obtained from total TIMP-1. We measured the uncomplexed fraction of TIMP-1, using a thoroughly validated ELISA specific for this fraction, in 341 tumor tissue extracts obtained from patients with primary breast cancer. These measurements were related to previously performed measurements of total TIMP-1 as well as to patient outcome. The observation time was 8.3 years (range, 7.3-11.3 years). During this period 136 patients died, and 153 patients experienced recurrence of disease. Cox regression analysis of recurrence-free survival (RFS) suggested that a score based on both uncomplexed and total TIMP-1, reflecting the tumor level of TIMP-1/MMP complexes, would be a more precise estimate of prognosis than total TIMP-1 alone. Univariate survival analysis showed a highly significant relationship between high values of the score and poor outcomes for RFS (p = 0.0002; hazard ratio = 2.7; 95% confidence interval, 1.5-4.8). Similar results were found for overall survival (p = 0.0001; hazard ratio = 3.3; 95% confidence interval, 1.8-6.3). Multivariate analysis of RFS and overall survival demonstrated that the score was significant including the classical prognostic factors used in breast cancer (p < 0.0001). The present study raises the hypothesis that it is the tumor level of TIMP-1/MMP complexes (i.e. activated matrix metalloproteinases) rather than TIMP-1 itself that determines prognosis, supporting the use of the combined score and not only total TIMP-1 in stratification of breast cancer patients.
Highlights
Several studies have demonstrated an association between high tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and a poor prognosis of primary breast cancer patients
It was demonstrated that the MAC 19 anti-TIMP-1 antibody used as detection antibody in the uncomplexed TIMP-1 ELISA detected the uncomplexed fraction of TIMP-1 as it did not recognize TIMP-1/matrix metalloproteinases (MMPs)-9 complexes (TIMP-1/ MMP-9 complexes generated signals at or below background in the specificity experiment)
The results from the present study demonstrate that inclusion of TIMP-1 in stratification of breast cancer patients cannot be based on measurements of total TIMP-1 alone as this is not the sole determinant of prognosis
Summary
Patients—Patient characteristics have been described previously [16, 31]. In brief, the present study included 341 patients who underwent surgery in Denmark in the period 1989 –1993 for histologically verified primary breast cancer. To determine the concentrations of uncomplexed TIMP-1 in individual tumor tissue samples a calibration curve was constructed for every assay plate by including recombinant free TIMP-1 in serial dilutions run in duplicate. Validation—The uncomplexed TIMP-1 ELISA was previously used for plasma samples [30] For this reason, it was necessary to validate the assay for measurements in tumor tissue samples. For determination of intra-assay variation, 32 identical samples of the tumor tissue extract pool diluted 1:101 in dilution buffer were measured in duplicate on the same plate. Interassay variation was determined by including duplicates of 1:101 dilutions of control tissue extract pool on every plate measured (n ϭ 11).
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