Sidse Ørnbjerg Würtz

Banking of Biological Fluids for Studies of Disease-associated Protein Biomarkers

With the increasing demand of providing personalized medicine the need for biobanking of biological material from individual patients has increased. Such samples are essential for molecular research aimed at characterizing diseases at several levels ranging from epidemiology and diagnostic and prognostic classification to prediction of response to therapy. Clinically validated biomarkers may provide information to be used for diagnosis, screening, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease), and prediction of response to treatment and as a surrogate response marker. Many types of biological fluids or tissues can be collected and stored in biorepositories. Samples of blood can be further processed into plasma and serum, and tissue pieces can be either frozen or fixed in formalin and then embedded into paraffin. The present review focuses on biological fluids, especially serum and plasma, intended for study of protein biomarkers. In biomarker studies the process from the decision to take a sample from an individual to the moment the sample is safely placed in the biobank consists of several phases including collection of samples, transport of the samples, and handling and storage of samples. Critical points in each step important for high quality biomarker studies are described in this review. Failure to develop and adhere to robust standardized protocols may have significant consequences as the quality of the material stored in the biobank as well as conclusions and clinical recommendations based on analysis of such material may be severely affected.

Biology and potential clinical implications of tissue inhibitor of metalloproteinases-1 in colorectal cancer treatment

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the industrialized world. About half of “curatively” resected patients develop recurrent disease within the next 3–5 years despite the lack of clinical, histological and biochemical evidence of remaining overt disease after resection of the primary tumour. Availability of validated biological markers for early detection, selection for adjuvant therapy, prediction of treatment efficacy and monitoring of treatment efficacy would most probably increase survival. Tissue inhibitor of metalloproteinases-1 (TIMP-1) may be such a marker. TIMP-1 inhibits the proteolytic activity of metalloproteinases, which are centrally involved in tumour invasion and metastases. However, in clinical investigations high tumour tissue or plasma levels of TIMP-1 have shown a strong and independent association with a shorter survival time in CRC patients, suggesting that TIMP-1 could have a tumour-promoting function. Furthermore, measurement of plasma TIMP-1 has been shown to be useful for disease detection, with a high sensitivity and high specificity for early-stage colon cancer. This review describes some basic information on the current knowledge of the biology of TIMP-1 as well as the potential use of TIMP-1 as a biological marker in the management of CRC patients.

Plasma and Serum Levels of Tissue Inhibitor of Metalloproteinases-1 Are Associated with Prognosis in Node-negative Breast Cancer A Prospective Study

The tumor level of TIMP-1 has been suggested as a new prognostic marker in breast cancer. The purpose of this study was to investigate whether TIMP-1 also carries prognostic information when measured in blood as this is a much more preferable material compared with tumor extracts. Using ELISA, TIMP-1 was measured in prospectively collected preoperative plasma and serum samples from 519 patients with primary breast cancer, and the measurements were related to patient outcome. The median age of the patients was 58 years (range, 38–80 years), and the median follow-up time was 1043 days (range, 300–1630 days). Plasma and serum TIMP-1 measurements correlated significantly with each other with a Pearson correlation coefficient of 0.75 (p < 0.0001). For univariate survival analysis, patients were divided into quartiles according to increasing TIMP-1 levels (Q1–Q4). Analysis of all patients showed that high TIMP-1 plasma levels were significantly associated with a shorter disease-free survival. Subgroup analysis showed that plasma TIMP-1 significantly predicted the prognosis of node-negative patients but not of node-positive patients. Importantly plasma TIMP-1 was able to further stratify low risk node-negative patients. High serum TIMP-1 levels were associated with a shorter disease-free survival; however, the association was not statistically significant. In contrast, serum TIMP-1 significantly predicted the prognosis of node-negative and low risk patients. In multivariate survival analysis of node-negative patients including all the classical prognostic parameters, plasma TIMP-1 remained significantly associated with prognosis when comparing Q1 with Q2 and Q4. Serum TIMP-1 remained significant when comparing Q1 with Q4. Taken together, this study is to our knowledge the first large prospective study suggesting that TIMP-1 carries independent prognostic information when measured in blood, especially plasma. This was especially true in the node-negative group of patients and in patients already defined as low risk patients using the currently available prognostic parameters.

A Phase 2, randomized, double-blind, placebo-controlled trial of clinical activity and safety of subcutaneous A6 in women with asymptomatic CA125 progression after first-line chemotherapy of epithelial ovarian cancer

OBJECTIVESnA6 is a novel peptide that interferes with single-chain urokinase plasminogen activator activity and has shown anti-angiogenic, anti-migratory, and anti-invasive properties. We evaluated clinical efficacy and safety of subcutaneously administered A6 in women with epithelial ovarian cancer.nnnMETHODSnWomen with epithelial ovarian, fallopian tube, or primary peritoneal cancer in clinical remission after first-line chemotherapy with 2 consecutive increases of CA125 values above normal but with no disease on physical examination or imaging studies were randomly assigned to receive daily subcutaneous injections of placebo, low-dose A6 (150 mg), or high-dose A6 (300 mg) until disease progression or end of study participation. Primary endpoints were time to clinical progression of disease and safety of A6. Secondary endpoints were changes in serum CA125 and biomarkers of the urokinase system.nnnRESULTSnData are available for 24 women (placebo, n=12; low-dose, n=8; high-dose n=4). A6 therapy was associated with a statistically significant delay in time to clinical progression (log-rank p-value 0.01) with a median of 100 days (95% CI: 64,168) for women who received A6 compared with 49 days (95% CI: 29,67) for women who received placebo. The treatments appeared to be well tolerated. Treatment was not associated with CA125 response (p=0.44). On-treatment values for plasma urokinase plasminogen activator receptor were statistically significantly lower in the A6 groups compared with placebo (p=0.02).nnnCONCLUSIONSnA6 therapy increases time to clinical disease progression and appears to be well tolerated in this patient population.

Measurement of the uncomplexed fraction of tissue inhibitor of metalloproteinases-1 in the prognostic evaluation of primary breast cancer patients

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.

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.

Association between tumor tissue TIMP-1 levels and objective response to first-line chemotherapy in metastatic breast cancer.

In a previous study from our laboratory, high tumor levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) have been associated with an adverse response to chemotherapy in metastatic breast cancer suggesting that TIMP-1, which is known to inhibit apoptosis, may be a new predictive marker in this disease. The purpose of this study was to investigate the association between TIMP-1 and objective response to chemotherapy in an independent patient population consisting of patients with metastatic breast cancer from Sweden and Denmark. TIMP-1 was measured using ELISA in 162 primary tumor extracts from patients who later developed metastatic breast cancer and these levels were related to the objective response to first-line chemotherapy. Increasing levels of TIMP-1 were associated with a decreasing probability of response to treatment, reaching borderline significance (ORxa0=xa01.59, 95% CI: 0.97–2.62, Pxa0=xa00.07). This OR is very similar to the result from our previous study. Increasing levels of TIMP-1 were also associated with a shorter disease-free survival and overall survival, however, not statistically significant. The results from the present study support previous data that TIMP-1 is associated with objective response to chemotherapy for metastatic breast cancer.

Plasma levels of the MMP-9:TIMP-1 complex as prognostic biomarker in breast cancer: a retrospective study

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; Pu2009=u20090.03 and Pu2009=u20090.028 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.

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.

Tissue inhibitor of metalloproteinases-1 protects the human breast carcinoma cell line MCF-7 S1 against anthracycline-induced cell death by activation of the akt survival pathway.

Abstract #2019 Wurtz S and Schrohl A contributed equally.u2028 Background. A large proportion of breast cancer patients are offered chemotherapy following primary surgery, however, many of these patients do not benefit from the treatment. Currently, only few markers exist to predict whether a breast cancer patient will respond to chemotherapy in general or to specific types of chemotherapy. Thus, additional predictive markers need to be identified to ensure a more effective treatment of the patients. In this regard, recent clinical studies performed in our laboratory have demonstrated that high tumor tissue levels of TIMP-1 are associated with a poor response to the most frequently used chemotherapeutic drugs (CMF or anthracyclines) (Schrohl et al., Clin. Cancer Res. 2006 and Klintman et al. manus. in prep.). The purpose of the present study was to investigate the molecular explanation for the clinical findings concerning the predictive value of TIMP-1 using an in vitro approach.u2028 Methods. We stably transfected the human breast carcinoma cell line MCF-7 S1 with the human TIMP-1 gene and established single cell clones expressing different levels of TIMP-1. We then compared the sensitivity of these cells to epirubicin and taxol using cell death and apoptosis assay. In addition, we investigated the signalling pathway that could be involved in the TIMP-1 mediated protection against treatment using western blotting of key molecules.u2028 Results. We found that clones expressing high levels of TIMP-1 were significantly less sensitive to epirubicin. In contrast, no difference in sensitivity to taxol was demonstrated between high and low TIMP-1 clones. When investigating the signalling pathway involved in the difference in sensitivity we found that phosphorylated Akt was highly up-regulated in the high TIMP-1 clones compared to the low TIMP-1 clones following chemotherapeutic treatment.u2028 Conclusion. TIMP-1 protects the MCF-7 S1 cells against anthracycline-induced cell death but not against taxol. Thus, TIMP-1 may be used to discriminate between patients likely to benefit from anthracyclines and patients who should be offered an alternative drug. Furthermore, we found that this protection is probably executed through counteraction of the apoptotic signal by activation of the Akt survival pathway. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2019.