High Tissue Inhibitor Of Metalloproteinases-1 Levels Research Articles

Tissue Inhibitor of Metalloproteinases-1 Overexpression Mediates Chemoresistance in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is among the most aggressive breast cancer subtypes. Despite being initially responsive to chemotherapy, patients develop drug-resistant and metastatic tumors. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a secreted protein with a tumor suppressor function due to its anti-proteolytic activity. Nevertheless, evidence indicates that TIMP-1 binds to the CD63 receptor and activates noncanonical oncogenic signaling in several cancers, but its role in mediating TNBC chemoresistance is still largely unexplored. Here, we show that mesenchymal-like TNBC cells express TIMP-1, whose levels are further increased in cells generated to be resistant to cisplatin (Cis-Pt-R) and doxorubicin (Dox-R). Moreover, public dataset analyses indicate that high TIMP-1 levels are associated with a worse prognosis in TNBC subjected to chemotherapy. Knock-down of TIMP-1 in both Cis-Pt-R and Dox-R cells reverses their resistance by inhibiting AKT activation. Consistently, TNBC cells exposed to recombinant TIMP-1 or TIMP-1-enriched media from chemoresistant cells, acquire resistance to both cisplatin and doxorubicin. Importantly, released TIMP-1 reassociates with plasma membrane by binding to CD63 and, in the absence of CD63 expression, TIMP-1-mediated chemoresistance is blocked. Thus, our results identify TIMP-1 as a new biomarker of TNBC chemoresistance and lay the groundwork for evaluating whether blockade of TIMP-1 signal is a viable treatment strategy.

TIMP-1 is Overexpressed and Secreted by Platinum Resistant Epithelial Ovarian Cancer Cells.

Epithelial Ovarian Cancer (EOC) is the most lethal gynecological cancer in developed countries, and the development of new strategies to overcome chemoresistance is an awaited clinical need. Angiogenesis, the development of new blood vessels from pre-existing vasculature, has been validated as a therapeutic target in this tumor type. The aim of this study is to verify if EOC cells with acquired resistance to platinum (PT) treatment display an altered angiogenic potential. Using a proteomic approach, we identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) as the only secreted factor whose expression was up-regulated in PT-resistant TOV-112D and OVSAHO EOC cells used as study models. We report that TIMP-1 acts as a double-edged sword in the EOC microenvironment, directly affecting the response to PT treatment on tumor cells and indirectly altering migration and proliferation of endothelial cells. Interestingly, we found that high TIMP-1 levels in stage III–IV EOC patients associate with decreased overall survival, especially if they were treated with PT or bevacizumab. Taken together, these results pinpoint TIMP-1 as a key molecule involved in the regulation of EOC PT-resistance and progression disclosing the possibility that it could be used as a new biomarker of PT-resistance and/or therapeutic target.

Abstract 1098: TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC)

Abstract BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Recent studies have demonstrated that TIMP-1 promotes tumor growth in an MMP-independent manner. We have previously shown that elevated plasma TIMP-1 predicts worse survival in mCRPC patients; however, the mechanism underpinning this association is unknown. Normal and malignant neuroendocrine (NE) cells, from various organs, express TIMP-1 as measured by immunohistochemistry (IHC). We hypothesized that elevated blood levels of TIMP-1 in mCRPC patients results from androgen deprivation therapy-induced NE differentiation. METHODS: In this study, we used qPCR, ELISA and IHC to correlate the expression of TIMP-1 and NE markers such as chromogranin A (CGA) in 7 prostate cancer cell lines, 37 patient serum samples and prostate cancer (adenocarcinoma and NE cancer) tissue microarrays. RESULTS: We first examined TIMP-1 and NE marker (NSE and PTHLH) mRNA expression in 7 prostate cancer cell lines. Our results show that PC-3, PC-3M, DU145 cells express high levels of TIMP-1 as well as NE markers. On the other hand, LNCAP, VCAP and LAPC-4 cells had barely detectable levels of TIMP-1 and much lower expression of NE markers. We then measured serum TIMP-1 and CGA levels from CRPC patient samples by ELISA. Patients with castration resistant disease had significantly higher serum TIMP-1 levels (mean value of 373.5 ng/ml) than patients with hormone sensitive disease (mean value of 304.8 ng/ml) (p=0.01), suggesting that castration resistance in prostate cancer patients is associated with increased TIMP-1 production. Interestingly, when we divided patients using a cutoff of 100 ng/ml (based on cutoffs reported in published literature), serum TIMP-1 was significantly higher in the high CGA group (p=0.018), indicating an association between TIMP-1 production and existing NE markers. We also compared PSA values between low and high CGA groups and found that the high CGA group was associated with lower PSA levels (p=0.029). Next, we explored TIMP-1 expression in NE tumor tissues. We performed TIMP-1 and CGA IHC staining in archival liver neuroendocrine and thyroid cancers and found that TIMP-1 expression was associated with CGA expression. In prostate cancer specimens, normal prostate epithelial cells expressed high levels of TIMP-1, but the expression was lost in adenocarcinomas. In contrast, prostatic neuroendocrine cancers expressed high levels of TIMP-1 as well as the NE marker CGA. CONCLUSIONS: These observations support the further evaluation of TIMP-1 as a tissue and serum biomarker for NE differentiation in CRPC. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Gong, Uma Chippada-Venkata, Xudong Dai, Matthew D. Galsky, Jiaoti Huang. TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2014-1098

Serum levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in human herpesvirus-6-infected infants with or without febrile seizures.

Human herpesvirus-6 (HHV-6) is a cause of exanthema subitum and, sometimes, of febrile seizures. However, the pathogenesis of febrile seizures associated with HHV-6 infection remains unclear. We investigated serum matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) levels in infants with HHV-6 infection. Serum levels of both MMP-9 and TIMP-1 were significantly higher in infants with HHV-6 infection than in controls. Serum TIMP-1 levels were significantly higher in infants with febrile seizures than in infants without febrile seizures. Serum MMP-9/TIMP-1 ratios were significantly lower in infants with febrile seizures than in infants without febrile seizures. In infants with HHV-6 infection, positive correlations were found between serum MMP-9 concentrations and the white blood cells (WBC) count, and between serum TIMP-1 concentrations and the WBC count. Positive correlations were also found between the amounts of HHV-6 DNA and the ratios of MMP-9/TIMP-1 in infants with HHV-6 infection. In conclusion, we suggest that high serum levels of MMP-9 and TIMP-1 in infants with HHV-6 infection may induce dysfunction of the blood-brain barrier, eventually causing febrile seizures.

Positron emission tomography/computed tomography for optimized colon cancer staging and follow up

Objectives. Optimal management of colon cancer (CC) requires detailed assessment of extent of disease. This study prospectively investigates the diagnostic accuracy of 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography (PET/CT) for staging and detection of recurrence in primary CC. Material and methods. PET/CT for preoperative staging was performed in 66 prospectively included patients with primary CC. Diagnostic accuracy for PET/CT and CT was analyzed. In addition to routine follow up, 42 stages I–III CC patients had postoperative PET/CT examinations every 6 months for 2 years. Serological levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), carcinoembryonic antigen, and liberated domain I of urokinase plasminogen activator receptor were analyzed. Results. Accuracy for tumor, nodal, and metastases staging by PET/CT were 82% (95% confidence interval [CI]: 70; 91), 66% (CI: 51; 78), and 89% (CI: 79; 96); for CT the accuracy was 77% (CI: 64; 87), 60% (CI: 46; 73), and 69% (CI: 57; 80). Cumulative relapse incidences for stages I–III CC at 6, 12, 18, and 24 months were 7.1% (CI: 0; 15); 14.3% (CI: 4; 25); 19% (CI: 7; 31), and 21.4% (CI: 9; 34). PET/CT diagnosed all relapses detected during the first 2 years. High preoperative TIMP-1 levels were associated with significant hazards toward risk of recurrence and shorter overall survival. Conclusions. This study indicates PET/CT as a valuable tool for staging and follow up in CC. TIMP-1 provided prognostic information potentially useful in selection of patients for intensive follow up.

TIMP-1 Increases Expression and Phosphorylation of Proteins Associated with Drug Resistance in Breast Cancer Cells

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.

TIMP1 overexpression mediates resistance of MCF-7 human breast cancer cells to fulvestrant and down-regulates progesterone receptor expression

High levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP1) are associated with poor prognosis, reduced response to chemotherapy, and, potentially, also poor response to endocrine therapy in breast cancer patients. Our objective was to further investigate the hypothesis that TIMP1 is associated with endocrine sensitivity. We established a panel of 11 MCF-7 subclones with a wide range of TIMP1 mRNA and protein expression levels. Cells with high expression of TIMP1 versus low TIMP1 displayed significantly reduced sensitivity to the antiestrogen fulvestrant (ICI 182,780, Faslodex®), while TIMP1 levels did not influence the sensitivity to 4-hydroxytamoxifen. An inverse correlation between expression of the progesterone receptor and TIMP1 was found, but TIMP1 levels did not correlate with estrogen receptor levels or growth-promoting effects of estrogen (estradiol, E2). Additionally, the effects of fulvestrant, 4-hydroxytamoxifen, or estrogen on estrogen receptor expression were not associated with TIMP1 levels. Gene expression analyses revealed associations between expression of TIMP1 and genes involved in metabolic pathways, epidermal growth factor receptor 1/cancer signaling pathways, and cell cycle. Gene and protein expression analyses showed no general defects in estrogen receptor signaling except from lack of progesterone receptor expression and estrogen inducibility in clones with high TIMP1. The present study suggests a relation between high expression level of TIMP1 and loss of progesterone receptor expression combined with fulvestrant resistance. Our findings in vitro may have clinical implications as the data suggest that high tumor levels of TIMP1 may be a predictive biomarker for reduced response to fulvestrant.

Plasma TIMP-1 in patients with metastatic colorectal cancer treated with first-line oxaliplatin-based therapy with or without cetuximab: Results from the Nordic VII study.

e14710 Background: Plasma Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) is a biomarker in patients with metastatic colorectal cancer (mCRC). TIMP-1 inhibits matrix metalloproteinases and has tumor promoting functions. High levels of TIMP-1 associate with reduced sensitivity to irinotecan-based but not oxaliplatin-based treatment in patients with mCRC. Based on our prior studies, we here tested the hypothesis that high pretreatment plasma TIMP-1 correlate with short overall survival (OS) and short progression-free survival (PFS) but not response rate (RR) in patients with mCRC included in the NORDIC VII Study in which patients received oxaliplatin-based therapy. Methods: In the NORDIC VII Study patients were randomized to: A) Nordic FLOX; B) Nordic FLOX + cetuximab or C) Nordic FLOX intermittently + cetuximab continuously. Plasma samples for TIMP-1 analysis were available from 426 patients. Plasma TIMP-1 was determined using the MAC15 antibody in-house validated kinetic ELISA. Results: The three study populations were comparable and not different from the total intention to treat population. The median pretreatment plasma TIMP-1 was 269 ng/mL (58-1318 ng/mL). KRAS mutated in 149 patients (35%). There was no interaction between treatment arms and pretreatment plasma TIMP-1 either for PFS (p = 0.16) or OS (p = 0.64). High pretreatment plasma TIMP-1 was associated with shorter PFS (HR=1.22, 1.07-1.39, p=0.003) and shorter OS (HR=1.55, 1.36-1.80, p<0.0001). RR did not correlate significantly to pretreatment plasma TIMP-1 (OR=1.16, 0.91-1.49, p=0.22). Multivariate analysis (TIMP-1, Köhne index, KRAS and BRAF status, CRP, CEA, age, gender) demonstrated that high pretreatment plasma TIMP-1 was not an independent biomarker of PFS (HR=1.06, 0.88-1.28, p=0.52) or OS (HR=1.19, 0.96-1.47, p=0.11). Conclusions: Plasma TIMP-1 is a prognostic biomarker in patients with mCRC treated with 1st line oxaliplatin-based therapy +/- cetuximab. A predictive effect of plasma TIMP-1 could only be estimated for cetuximab treatment, and no significant treatment by marker interaction was observed.

Relationships between TIMP-1, CAIX, and clinical outcomes in Egyptian breast cancer

Serum levels of carbonic anhydrase IX (CAIX) and the tissue inhibitor of metalloproteinase 1 (TIMP-1) have been demonstrated to be associated with clinical outcomes. Elevated TIMP-1 levels have been associated with a poor prognosis of breast cancer. Therefore, this study was performed to explore the relationships between serum CAIX, TIMP-1, and clinical outcomes. One hundred and five female breast cancer patients, who do not receive adjuvant therapy at Mansoura University Hospital were recruited with informed consent for this study. Preoperative serum levels of CAIX and TIMP-1 were measured using an enzyme-linked immunosorbent assay. Among the breast cancer patients, 16.1 and 20.9 % had elevated TIMP-1 and CAIX l levels, respectively. In addition, 13.3 % had elevated both CAIX and TIMP-1 levels. Elevated serum levels CAIX was significantly associated with advanced histological grade, estrogen receptor status and progesterone receptor status. Moreover, Elevated TIMP-1 was significantly associated with number of tumor-positive lymph nodes and advanced histological grade. In Kaplan–Meier analysis, the disease-free survival (DFS) was lower in patients with high serum levels of CAIX and TIMP-1 compared to patients with normal serum concentration of CAIX and TIMP-1. Including TIMP-1, CAIX, and established prognostic factors in the multivariate analysis, the presence of elevated CAIX and TIMP-1 levels remained independent predictors of overall survival time (OS). A high serum TIMP-1 and CAIX levels were significantly associated with poorer DFS and OS among Egyptian primary breast cancer patients with elevated serum levels of CAIX and TIMP-1.

Plasma levels of TIMP-1 in chemo-naive patients with metastatic colorectal cancer treated with first-line FLOX with or without cetuximab: Results from the Nordic VII Study.

392 Background: KRAS status is presently the best known biomarker to select patients with metastatic colorectal cancer (mCRC) for therapy with EGFR inhibition. However, in NORDIC VII a survival benefit of adding cetuximab to the Nordic FLOX regimen could not be confirmed. Plasma Tissue Inhibitor of MetalloProteinases-1 (TIMP-1) is a promising biomarker in patients with mCRC, and has multiple actions including regulation of apoptosis, angiogenesis, cell growth and differentiation. High levels of TIMP-1 associate with reduced sensitivity to irinotecan-based treatment in patients with mCRC. We investigated whether high plasma TIMP-1 also correlates with response rate, progression-free survival (PFS) and overall survival (OS) in patients with mCRC treated in NORDIC VII. Methods: In NORCIC VII 571 patients with mCRC were randomized to: (A) Nordic FLOX; (B) Nordic FLOX + cetuximab or (C) Nordic FLOX intermittently + cetuximab continuously. Baseline plasma samples for TIMP-1 analysis were available from 426 patients (79%). Plasma TIMP-1 was determined using the MAC15 antibody in-house validated kinetic Enzyme Linked Immunosorbent Assay. Results: Median plasma TIMP-1 was 296 ng/mL. The tumor was KRAS mutated in 149 patients (35%). Best response did not correlate to TIMP-1 values (OR=1.16, 0.91-1.49, p=0.22). High plasma TIMP-1 was associated with short PFS (HR=1.22, 1.07-1.39, p=0.003) and OS (HR=1.55, 1.36-1.80, p<0.0001). Multivariate analysis (TIMP-1, age, gender, KRAS, ALP, WBC, platelets, CRP, CEA, WHO PS, no. of metastatic sites) demonstrated that high plasma TIMP-1 was not an independent biomarker of PFS (HR=0.95, 0.76-1.18, p=0.65) or OS (HR=1.17, 0.91-1.50, p=0.22). Multivariate analysis retained CRP, PS and no. of metastatic sites in the final model for PFS and alkaline phosphates, PS and no. of metastatic sites for OS. Conclusions: Plasma TIMP-1 is a new prognostic biomarker in patients with mCRC treated with 1st line Nordic FLOX +/- cetuximab. Due to the design of NORDIC VII, a predictive effect of plasma TIMP-1 could only be estimated for cetuximab treatment and no significant treatment by marker intervention was observed.

High expression of TIMP-1 in human breast cancer tissues is a predictive of resistance to paclitaxel-based chemotherapy

For breast cancer patients with lymph node metastasis, paclitaxel is the first-line chemotherapy drug. Clinical studies showed that some patients with breast cancer were insensitive to paclitaxel, which led to chemotherapy failure. Today, no validated markers exist for the prediction of chemotherapy sensitivity in this patient group. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to protect against apoptosis. Epidemiological studies have also associated elevated tumor tissue TIMP-1 levels with a poor response to cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemotherapy. Additionally, our previous study proved that TIMP-1 significantly decreased the sensitivity of breast cancer cells to paclitaxel-induced apoptosis by enhancing degradation of cyclin B1. These data imply that TIMP-1 may be a useful predictive biomarker for chemotherapy resistance. In this retrospective study, we investigated the association between expression levels of TIMP-1 protein in the primary tumor and objective response to paclitaxel-based chemotherapy in 99 patients with breast cancer. With Kaplan-Meier survival analysis, the patients with high TIMP-1 levels were found to have significantly worse 5-year DFS (71.1 %) than the patients with low levels (88.5 %; P = 0.020). Similarly, the patients with high TIMP-1 levels had significantly worse 5-year OS (78.9 %) than patients with low levels (96.7 %; P = 0.004). In Cox's univariate and multivariate analyses, TIMP-1 was prognostic for both DFS and OS. Our data showed that elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to paclitaxel-based chemotherapy, and TIMP-1 might be a potential biomarker for predicting response of breast cancer patients to paclitaxel-based chemotherapy.

P4-01-17: TIMP-1 Over-Expression Confers Resistance of MCF-7 Breast Cancer Cells to Fulvestrant.

Abstract Background Endocrine resistance represents a major challenge in the management of estrogen receptor (ER) positive breast cancer. Currently no predictive biomarkers for endocrine resistance in ERpositive breast cancer patients are in clinical use. In a clinical study, patients with metastatic breast cancer and high levels of serum Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) had less benefit from endocrine therapy than patients with a lower level of serum TIMP-1 [1]. Therefore, we evaluated the association between TIMP-1 and response to endocrine therapy using an in vitro approach. We have previously presented initial results on TIMP-1 and response to endocrine therapy [2]. Materials and Methods: MCF-7 cells were stably transfected with pcDNA3.1(Hyg)-TIMP-1 plasmid, and a panel of 11 subclones with different expression levels of TIMP-1 was generated. TIMP-1 expression levels were confirmed using enzyme-linked immunosorbent assay (ELISA). Four subclones with high or low TIMP-1 expression were analyzed for the growth response to estrogen, 4-hydroxytamoxifen and fulvestrant. These four subclones were analyzed for protein expression by western blotting. All subclones were analyzed by whole human genome oligo microarrays 4×44K for determination of gene expression levels. Data were analyzed using the limma R/Bioconductor package. Paired-end Solexa sequencing was applied to selected subclones with high and low TIMP-1 levels to identify transcriptomic changes. Results: High expression of TIMP-1 was associated with resistance to fulvestrant, whereas growth response to either estrogen or 4-hydroxytamoxifen was independent of TIMP-1 expression levels. High expression of TIMP-1 protein and mRNA was associated with undetectable levels of progesterone receptor (PgR) protein and mRNA whereas ER protein and mRNA levels were unaffected by TIMP-1. To characterize the potential role of TIMP-1 in estrogen signaling we analyzed the expression of reported estrogen-responsive genes and no general change was observed. We identified genes that correlated positively or negatively to TIMP-1 expression. Among the identified genes was PgR, which is a direct target for ER. Conclusion: Our data suggest that a high expression of TIMP-1 in vitro is associated with resistance to fulvestrant but not to 4-hydroxytamoxifen. Estrogen-regulated genes are not generally affected by changes in TIMP-1 expression levels and therefore TIMP-1 appears to affect endocrine resistance through other mechanisms than globally regulating ER signaling. However, high expression of TIMP-1 is associated with loss of PgR and this may be related to the resistance towards fulvestrant.

Serum TIMP-1 Predicts Survival Outcomes of Invasive Breast Carcinoma Patients: A Meta-analysis

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a small secretory glycoprotein with multifunctional activity including anti-apoptosis and the inhibition of matrix metalloproteinase in invasive breast carcinomas. There have been contradictory results as to whether TIMP-1 is a poor or good prognostic factor in breast cancer patients. To address this controversy, we conducted a meta-analysis for the relationship between TIMP-1 levels and prognostic parameters in the breast cancer. The relevant published studies were pooled according to the defined selection criteria. The effect sizes of overall survival and prognostic parameters were calculated by a hazard ratio (HR) or an odds ratio (OR). HRs or ORs were combined using a random-effects model. Survival outcomes between high or elevated and low or normal serum TIMP-1 levels were compared by uni- and multivariate analyses involving 886 and 844 breast cancer patients, respectively. Patients with high or elevated serum TIMP-1 levels had unfavorable survival outcomes compared to patients with low or normal serum TIMP-1 levels in the uni- and multivariate analyses (HR, 1.7 and 2.4; p <0.001 and p= 0.033, respectively). However, no survival difference was evident in the data from tissue TIMP-1 levels by enzyme-linked immunosorbent assay and the expression of tissue TIMP-1 mRNA. The high or positive immunohistochemical expression of tissue TIMP-1 protein was not related to adjusted and unadjusted HRs, lymph node metastasis, and clinical stages. This meta-analysis indicates that serum TIMP-1 levels may be useful for predicting survival outcomes of invasive breast cancer patients.

Plasma TIMP-1 levels and treatment outcome in patients treated with XELOX for metastatic colorectal cancer

BackgroundThe aim was to evaluate the association between plasma tissue inhibitor of metalloproteinase-1 (TIMP-1) and serum carcinoembryonic antigen (CEA) levels and outcome in patients with metastatic colorectal cancer (mCRC) receiving XELOX (combination chemotherapy with capecitabine and oxaliplatin) as first-line treatment. Patients and methodsOne hundred and twenty patients were included. Blood samples were collected before treatment and 3 weeks later before the next treatment cycle. Plasma TIMP-1 and serum CEA levels were correlated to treatment outcome. ResultsNo significant associations between baseline TIMP-1 or CEA levels and best response to treatment or progression-free survival (PFS) could be demonstrated. In contrast, high baseline plasma TIMP-1 levels were associated with poor overall survival (OS), P=0.008, hazard ratio (HR)=1.80 [95% confidence interval (CI): 1.17–2.78]. Furthermore, increase in TIMP-1 levels from baseline to immediately before the second cycle of chemotherapy had a significant negative effect on survival (P=0.03, HR=1.30, 95% CI: 1.02–1.65) while a decrease in TIMP-1 was significantly associated with a higher objective response rate (P=0.03). ConclusionsBoth high baseline and subsequent increase in TIMP-1 levels were associated with shorter OS in patients with mCRC receiving XELOX as first-line treatment, whereas baseline TIMP-1 levels were not associated with response or PFS following XELOX treatment.

Abstract P4-07-04: Preoperative Plasma Concentration of MMP-9/TIMP-1 Complexes Is Not Associated with Disease Outcome in Primary Breast Cancer (N=483)

Abstract 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&amp;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 (&amp;gt; 70 years) and hormone receptor status contributed significantly to the model for RFS (P&amp;lt;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.

Preoperative plasma TIMP-1 is an independent prognostic indicator in patients with primary colorectal cancer: A prospective validation study

Background Previous studies have suggested plasma tissue inhibitor of metalloproteinases-1 (TIMP-1) as a stage independent prognostic marker in colorectal cancer (CRC) patients. The aim was to validate plasma TIMP-1 and serum carcino-embryonic antigen (CEA) levels as prognostic indicators in an independent population-based cohort of patients with CRC. Patients and methods During 2000–2003, plasma and serum were collected preoperatively from 322 patients treated for primary CRC. TIMP-1 and CEA levels were determined by validated ELISA platforms. Results High TIMP-1 and CEA levels each associated with poor overall survival (OS); TIMP-1 (hazard ratio (HR) 2.1; 95% confidence interval (CI) 1.6–2.7) and CEA (HR 1.2; 95% CI 1.1–1.3), and disease-free survival (DFS); TIMP-1 (HR 2.0; 95% CI: 1.5–2.6) and CEA (HR 1.2; 95% CI: 1.1–1.4) in univariate analyses. In stratified analyses of stages II and III, TIMP-1 levels associated significantly with OS and DFS in stages II and III, associations were not found for CEA. Multivariate analysis for OS, including TIMP-1 and CEA levels and clinico-pathological baseline variables, revealed significant association of TIMP-1 (HR 1.8; 95% CI 1.3–2.4) but not CEA levels. Conclusions This independent prospective validation study confirms the significant association between preoperative plasma TIMP-1 levels and survival of CRC patients: TIMP-1 provided stronger prognostic information than CEA. Thus, this study brings plasma TIMP-1 to the next level of evidence for its clinical use as a prognostic marker in CRC patients.

Primary tumor tissue levels of TIMP-1 and outcome following chemotherapy with docetaxel monotherapy or docetaxel in combination with gemcitabine in patients with locally advanced or metastatic breast cancer.

1079 Background: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is recognized to protect against apoptosis, and previous studies have demonstrated that high levels of TIMP-1 in primary tumor tissue are associated with poor response to anthracycline-containing chemotherapy in adjuvant as well as metastatic treatment of breast cancer (Schrohl et al., Clin. Cancer Res. 2006; Klintman et al. Breast Cancer Res. Treat. 2009; Schrohl et al., BMC Cancer 2009; Willemoe et al., EJC 2009). The purpose of the present study was to evaluate whether TIMP-1 immunoreactivity also predicts sensitivity/resistance to taxanes. Methods: Tissue micro arrays from the primary tumor tissue from 260 patients with locally advanced or metastatic breast cancer randomly assigned to docetaxel or docetaxel plus gemcitabine were included. Assessment of TIMP- 1 status was performed using immunohistochemistry and tumor samples were regarded as positive if epithelial breast cancer cells were stained by the anti-TIMP-1 monoclonal antibody VT7 (S∅rensen et al., J. Hist. Cytochem. 2006). Relationships between the effects of chemotherapy and TIMP-1 status were analyzed using univariate analysis. Endpoints were best clinical response (BCR, not confirmed) and progression-free survival (PFS). Results: Since there were no differences in BCR or PFS between the two randomization arms, the patient cohort was pooled. 150 (58%) of the tumors were classified as tumor cell TIMP-1 positive. Among TIMP-1 positive patients 46 (31%) obtained complete response (CR) or partial response (PR), while 39 (35%) of the TIMP-1 negative patients had CR or PR (Fisher's exact test, p= 0.43). Applying PFS as end-point no significant difference between TIMP-1 negative versus TIMP-1 positive patients was observed (log-rank test, p= 0.29). Conclusions: The present study is the first to demonstrate that TIMP-1, a marker of anthracycline resistance, is not associated with a poor response to docetaxel in patients with locally advanced or metastatic breast cancer. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Patent application on TIMP-1 as a predictive biomarker

Examination of TIMP-1 Levels and Relapse-Free Survival for Patients in NCIC CTG MA.14 Who Received Adjuvant Tamoxifen +/- Octreotide LAR.

Abstract Background: Tissue inhibitor of metalloproteinase-1 (TIMP-1) has been shown to have diverse multifunctional roles in tumorigenesis. AL/KL/SA postulated from first line metastatic endocrine therapy that elevated pre-treatment serum TIMP-1 required additional therapy. NCIC CTG MA.14 adjuvant endocrine trial permitted examination of whether TIMP-1 serum levels post-chemotherapy was associated with RFS. Methods: NCIC CTG MA.14 is a trial where 667 postmenopausal patients were randomized to receive adjuvant tamoxifen +/- octreotide LAR with a stratification factor of no, concurrent or sequential chemotherapy. For the purposes of this work, this was simplified to whether a patient had or had not received adjuvant chemotherapy prior to the serum draw. Serum TIMP-1 was assessed on &amp;gt;90% of the trial patients. We examined the effect of baseline TIMP-1 levels on relapse-free survival (RFS) of 1) all types, 2) bone only, 3) all types of bone, and 4) non-bone, by timing of chemotherapy before the serum draw utilizing continuous TIMP (ng/ml) and categorical TIMP (&amp;lt;454, &amp;gt;454 ng/ml, based on 95% non-parametric cut-point for healthy post-menopausal females). Data were available on patient and tumour characteristics of age (years), pathologic tumour size (cm), pathologic lymph node status (# nodes), IGF-1, C-peptide, IGFBP-3. Step-wise forward Cox multivariate models were used where a factor was added if p&amp;lt;=0.05, and interaction terms were added for TIMP-1 level and timing of chemotherapy. Results: High (categorical) TIMP-1 was significantly associated with longer RFS (p=0.04) in the non-bone RFS multivariate model in the subgroup of patients who had prior chemotherapy, but not in those who did not. The interaction between TIMP-1 and administration of chemotherapy before serum draw was significant (p=0.02). High (continuous) TIMP-1 was significantly associated with longer bone only RFS (p=0.04) in subgroup who had no chemotherapy before the serum draw, but not in those who may or may not have had it after. There was no significant interaction effect. Categorical and continuous TIMP-1 were not associated with any other type of RFS. Conclusions: We found a predictive benefit with the assessment of serum TIMP-1 in hormone receptor positive patients, that high TIMP-1 levels after adjuvant chemotherapy were predictive of a reduction in non-bone RFS. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3022.

Tumor tissue levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and outcome following adjuvant chemotherapy in premenopausal lymph node-positive breast cancer patients: A retrospective study

BackgroundWe have previously demonstrated that high tumor tissue levels of TIMP-1 are associated with no or limited clinical benefit from chemotherapy with CMF and anthracyclines in metastatic breast cancer patients. Here, we extend our investigations to the adjuvant setting studying outcome after adjuvant chemotherapy in premenopausal lymph node-positive patients. We hypothesize that TIMP-1 high tumors are less sensitive to chemotherapy and accordingly that high tumor tissue levels are associated with shorter survival.MethodsFrom our original retrospectively collected tumor samples we selected a group of 525 pre-menopausal lymph node-positive patients (adjuvant treatment: CMF, 324 patients; anthracycline-based, 99 patients; no adjuvant chemotherapy, 102 patients). TIMP-1 levels were measured using ELISA in cytosolic extracts of frozen primary tumors. TIMP-1 was analyzed as a continuous variable and as a dichotomized one using the median TIMP-1 concentration as a cut point between high and low TIMP-1 groups. We analyzed the benefit of adjuvant CMF and anthracyclines in univariate and multivariable survival models; endpoints were disease-free (DFS) and overall survival (OS).ResultsIn this selected cohort of high-risk patients, and in the subgroup of patients receiving no adjuvant therapy, TIMP-1 was not associated with prognosis. In the subgroup of patients treated with anthracyclines, when analyzed as a continuous variable we observed a tendency for increasing TIMP-1 levels to be associated with shorter DFS (multivariable analysis, HR 1.75, 95% CI 1.00-3.07, P = 0.05) and a significant association between increasing TIMP-1 and shorter OS in both univariate (HR 3.52, 95% CI 1.54-8.06, P = 0.003) and multivariable analyses (HR 4.19, 95% CI 1.67-10.51, P = 0.002). No statistically significant association between TIMP-1 and DFS was observed in the CMF-treated patients although high TIMP-1 was associated with shorter OS when analyzed as a dichotomized variable (HR 1.64, 95% CI 1.02-2.65, P = 0.04).ConclusionIn the subgroup of patients receiving adjuvant chemotherapy we found an association between shorter survival after treatment in TIMP-1 high patients compared with TIMP-1 low patients, especially in patients receiving anthracycline-based therapy. This suggests that high tumor tissue levels of TIMP-1 might be associated with reduced benefit from classical adjuvant chemotherapy. Our findings should be validated in larger prospective studies.

Correlation of plasma TIMP-1 levels and disease-free survival in primary colorectal cancer independent of serum CEA levels

e22040 Background: Introduction of independent prognostic markers may play a significant role in future treatment of early stage colorectal cancer (CRC). CEA is still the only recommended (ASCO and EGTM) serological marker in CRC. However, Tissue Inhibitor of Metallo Proteinases-1 (TIMP-1), which is a glycoprotein that acts as an inhibitor of most of the active matrix metalloproteinases, has previously been shown to carry independent prognostic information in patients with primary CRC. The purpose of the present study was to assess the combination of preoperative serum CEA and plasma TIMP-1as prognostic markers in patients undergoing resection for primary CRC. Methods: In the present prospective study serum and plasma samples were collected before surgery from 422 patients with primary CRC stage I-III. CEA was determined in serum by a commercial assay and TIMP-1 was determined in plasma using a thoroughly validated, in-house ELISA. Time to recurrence or death of CRC was registered and the association to serum CEA and plasma TIMP-1 levels were studied in a Cox multivariate model including age, gender, disease stage and tumor location. Hazard ratios and 95% confidence intervals (HR (95%CI)) for disease free survival (DFS) were calculated. Results: An event was recorded in 186 patients, 75 had local recurrence, 103 had a distant metastases (28 of these patients had both local recurrence and distant metastases) and 36 died from their cancer without a registered recurrence. Scoring CEA and TIMP-1 as continuous variables on a logarithmic scale (base 2), both serum CEA and plasma TIMP-1 were statistically significant in a multivariable analysis with HR=1.12 (1.03–1.21) and HR=1.51 (1.12–2.04), respectively. Additional calculations of low CEA plus low TIMP-1, high CEA plus low TIMP-1, low CEA plus high TIMP-1 and high CEA plus high TIMP-1 showed that high plasma TIMP-1 levels carried prominent information of poor prognosis independent of CEA. Conclusions: Preoperative serum CEA and plasma TIMP-1 levels were independent predictors of disease free survival for patients with primary CRC. Combination of the two proteins showed that TIMP-1 was a prominent predictor of poor DFS independent of CEA. [Table: see text]