Whether patients diagnosed with primary breast cancer are offered adjuvant systemic therapy following surgical removal of the tumor is based on prognosis. Prognosis is estimated in every patient using established prognostic variables. Unfortunately, when using the currently available prognostic parameters a significant proportion of patients are over-treated. Thus, in order to improve stratification of breast cancer patients, additional prognostic factors need to be identified. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of the promising candidates for new prognostic markers in breast cancer, as a number of studies have demonstrated an association between high tumor-tissue levels of TIMP-1 mRNA as well as TIMP-1 protein and a poor prognosis of breast cancer patients. TIMP-1 is a member of the TIMP family, currently comprising four members (TIMP-1-4), and its main function is inhibition of the activity of various matrix metalloproteinases (MMPs). The association between high levels of protease inhibitor and poor prognosis may be somewhat surprising, as proteolytic activity plays a pivotal role in cancer cell invasion and metastasis. However, the recent discovery of other biological functions of TIMP-1 such as growth-stimulating functions, as well as anti-apoptotic and pro-angiogenetic effects, may in part explain this paradox. The purpose of this review is to give an update on the current status of TIMP-1 in breast cancer, emphasizing the prognostic utility of the inhibitor. In addition, the suggested tumor-stimulatory roles of TIMP-1 will be outlined.

Background: Human epidermal growth factor receptor (HER-) 2 belongs to the family of epidermal growth factor receptors (EGFRs) with homology to HER1, HER3 and HER4. HER2 is over-expressed in approximately 20% of invasive human breast cancers (Wolff, Hammond et al. 2007). Over-expression of HER2 is associated with a poor prognosis; however, the protein can be targeted by anti-HER2 therapy (trastuzumab, Herceptin®) (Slamon, Leyland-Jones et al. 2001). Trastuzumab therapy is offered only to patients whose tumors over-express HER2 protein and/or show amplification of the HER2 gene. Accordingly, the expression level of HER2 protein and/or amplification of the HER2 gene are determined routinely in all newly diagnosed breast cancers. Essential to this testing of HER2 expression at the protein level is the availability of specific antibody-based test systems. Aim: The aim of the present study was to investigate the specificity of three clinically approved commercially available anti-HER2 antibodies towards members of the EGFR-family. Methods: We studied the antibody used in the Herceptest™ (Dako), the PATHWAY® antibody (Ventana Medical Systems, Inc.) and the Oracle™ antibody (Leica Microsystems). Antibody specificity was investigated by manually performed immunohistochemistry (IHC) and in competitive ELISAs. For IHC, Chinese Hamster Ovary (CHO) cells were transiently transfected with the intracellular domain of respectively HER1, HER2, HER3 and HER4 and all three antibodies were applied to sections of formalin-fixed paraffin-embedded (FFPE) transfected cells. In ELISA, cross reactivity towards HER1 and HER4 was tested with peptides corresponding to the C-terminal part of HER1 and HER4. Results: In IHC experiments, all three antibodies stained cells transfected with HER2. Binding of the antibodies to HER2 was confirmed bycompetitive ELISA. However, in IHC experiments the PATHWAY® and the Oracle™ antibodies also stained cells transfected with HER4. Competitive ELISAs confirmed binding of the PATHWAY® antibody to HER4, whereas binding of the Oracle™ antibody to HER4 could not be confirmed in a competitive ELISA. None of the antibodies cross reacted with HER1 and HER3 homologous to the HER2 binding site of the antibodies. Conclusions: Two out of three clinically validated anti-HER2 antibodies were shown to cross react with HER4 in FFPE cells. As determination of HER2 over-expression and/or amplification guides therapy with trastuzumab, a valid test result by the use of specific antibodies is crucial to ensure proper personalized therapy with HER2-targeted therapy. These results warrant further investigation of anti-HER2 antibodies and of the procedures for clinical determination of HER2 protein expression. References: Slamon DJ et al. (2001) J Med 344:783 Wolff AC et al. (2007) J Clin Oncol 25:118 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-08.

Background: Plasma levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) have been reported as predictors of poor prognosis. Indeed, in a previous study of 519 primary breast cancer patients we reported that high levels of TIMP-1 were associated with a poor prognosis (Würtz et al, 2008). In our previous study we quantified total levels of TIMP-1 in plasma; however, TIMP-1 is present in plasma in a non-complexed form and bound to various proteins. Thus, studying the different fractions might refine prognostic stratification and provide further insight into the role of TIMP-1 in tumor biology. Matrix Metalloproteinase-9 (MMP-9) is an important TIMP-1 binding partner and MMP-9 has previously been suggested as a breast cancer biomarker (Li et al, 2004; Somiari et al, 2006). Aim: The aim of the study was to analyze the concentration of MMP-9/TIMP-1 complexes in plasma from our previously studied primary breast cancer patients and evaluate whether these levels are associated with disease outcome. Materials and methods: Plasma concentrations of MMP-9/TIMP-1 complexes were measured using the human MMP-9/TIMP-1 Complex DuoSet® ELISA Development System (R&D Systems, Inc.) The ELISA was thoroughly validated for measurements of MMP-9/TIMP-1 complexes in EDTA plasma. Samples included preoperatively obtained EDTA plasma from consecutively enrolled patients with primary breast cancer. Of the previously studied 519 patients, 483 had plasma samples available for analysis and follow-up data registered by the Danish Breast Cancer Cooperative Group and were included in the present study. The median follow-up time was 5.1 years. The relationship between MMP-9/TIMP-1 complexes and classical prognostic parameters was analyzed and the association with recurrence-free survival (RFS; includes breast cancer relapse, contralateral breast cancer, other malignant disease, and death without a previous relapse) was studied. Results: The ELISA was validated with acceptable results. The median TIMP-1 concentration was 2.06 ng/mL. For analysis, patients were grouped in quartiles of increasing MMP-9/TIMP-1 plasma concentrations. MMP-9/TIMP-1 complex levels were associated with menopausal status and with hormone receptor status; no significant associations with other clinico-pathological parameters (tumor size, nodal status, malignancy grade, age) were found. No statistically significant difference in survival was observed among TIMP-1 high and low groups (quartiles, log-rank analysis p=0.96). In a Cox multivariable analysis (including tumor size, nodal status, hormone receptor status, malignancy grade, menopausal status, age), only age (> 70 years) and hormone receptor status contributed significantly to the model for RFS (P<0.001). MMP-9/TIMP-1 complex concentration was not associated with RFS in this model. Conclusions: In this group of primary breast cancer patients we did notfind any association between MMP-9/TIMP-1 complex levels in plasma and RFS. Total TIMP-1 levels have previously been shown to correlate with prognosis in this patient cohort. Accordingly, our current results point to other TIMP-1 fractions, i.e. the fraction of free TIMP-1 or TIMP-1 in complex with other plasma proteins, as potential indicators of poor prognosis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-04.