Breast Neoplasms: Brünner N

Sturgeon CM, Duffy MJ, Stenman UH, Lilja H, Brünner N, Chan DW, Babaian R, Bast RC, Dowell B, Esteva FJ, Haglund C, Harbeck N, Hayes DF, Holten-Andersen M, Klee GG, Lamerz R, Looijenga LH, Molina R, Nielsen HJ, Rittenhouse H, Semjonow A, Shih IeM, Sibley P, Sölétormos G, Stephan C, Sokoll L, Hoffman BR, Diamandis EP, Anonymous00039. · Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Edinburgh, UK. · Clin Chem. · Pubmed #19042984 No free full text.

Abstract: BACKGROUND: Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS: Published reports relevant to use of tumor markers for 5 cancer sites--testicular, prostate, colorectal, breast, and ovarian--were critically reviewed. RESULTS: For testicular cancer, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. alpha-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is <10 microg/L. In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, postoperative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing may be used for screening asymptomatic adults 50 years or older. For breast cancer, estrogen and progesterone receptors are mandatory for predicting response to hormone therapy, human epidermal growth factor receptor-2 measurement is mandatory for predicting response to trastuzumab, and urokinase plasminogen activator/plasminogen activator inhibitor 1 may be used for determining prognosis in lymph node-negative patients. CA15-3/BR27-29 or carcinoembryonic antigen may be used for therapy monitoring in advanced disease. CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in postmenopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer. CONCLUSIONS: Implementation of these recommendations should encourage optimal use of tumor markers.

Würtz SO, Schrohl AS, Mouridsen H, Brünner N. · Department of Veterinary Pathobiology, Ridebanevej 9, University of Copenhagen, Faculty of Life Sciences, Frederiksberg C, Denmark. · Acta Oncol. · Pubmed #18465326 No free full text.

Abstract: Improvement of the management of breast cancer patients has high priority. In this regard, prognostic stratification needs to be improved in order to ensure proper medical treatment of all patients and furthermore predictors of response to chemotherapy are urgently needed. As new treatment opportunities emerge in the future this need will continue to grow. Thus, the search for molecular markers of prognosis and prediction is ongoing. Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested as a marker of both prognosis and response to treatment. Several studies have demonstrated the association between TIMP-1 and prognosis in breast cancer and new studies within this area have focused on the possibility of using blood samples or paraffin embedded tissue instead of tumor tissue extracts for measurements of TIMP-1. Interestingly, recent studies have investigated the association between TIMP-1 and response to treatment showing that TIMP-1 may also carry predictive information on response to treatment. In this regard, results from studies of the molecular functions of TIMP-1 point to a role of TIMP-1 in the inhibition of tumor cell apoptosis as an explanation for the clinical findings. This review gives an update on the ongoing investigation of the potential role of TIMP-1 as a tumor marker in breast cancer. Furthermore, we link the clinical findings with studies of the molecular actions of the TIMP-1 protein, raising hypotheses that may explain why TIMP-1 could play an important role in future management of breast cancer patients.

Würtz SØ, Schrohl AS, Sørensen NM, Lademann U, Christensen IJ, Mouridsen H, Brünner N. · The Royal Veterinary and Agricultural University, Department of Veterinary Pathobiology, Ridebanevej 9, DK-1870 Frederiksberg C, Denmark. · Endocr Relat Cancer. · Pubmed #15947098 links to  free full text

Abstract: 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.

Mouridsen HT, Brünner N. · Danish Breast Cancer Cooperative Group, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. · Mol Cell Proteomics. · Pubmed #14762216 links to  free full text

Abstract: The Danish Breast Cancer Cooperative Group (DBCG) was established in 1977 with the aim to ensure optimal breast cancer diagnostics and therapeutic modalities on a nationwide basis. DBCG was organized in such a way so it represents a broad interdisciplinary collaboration with established clinical databases and biobanks. This review summarizes the infrastructures, such as those of the DBCG, that are required to facilitate translational research studies aiming at further diagnostic and therapeutic improvements through interactions directed at prevention, early diagnosis, and treatment of primary breast cancer.

Brünner N, Nielsen HJ, Hamers M, Christensen IJ, Thorlacius-Ussing O, Stephens RW. · Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark. · APMIS. · Pubmed #10190293 No free full text.

Abstract: The cell surface plasminogen activation system functions in promoting tumor dissemination, and is facilitated by a glycolipid anchored three domain receptor for urokinase. This receptor can also be found in a soluble form (suPAR) in extracts of tumors, as well as in plasma from both healthy individuals and cancer patients. The suPAR in plasma consists of the intact three domain protein, but neither the precise mechanism of its release from cell surfaces, nor its biological function are understood. Increased levels of plasma suPAR have been found in patients with cancers of the lung, breast, ovary, and colon, and recent data now indicates that the level of the molecule is related to patient prognosis.

Usher PA, Sieuwerts AM, Bartels A, Lademann U, Nielsen HJ, Holten-Andersen L, Foekens JA, Brünner N, Offenberg H. · Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Ridebanevej 9, 1870 Frederiksberg C, Denmark. · Mol Oncol. · Pubmed #19383295 No free full text.

Abstract: TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP-1 may hold important clinical information, and either alone or in combination with measurement of full-length TIMP-1 they may improve the prognostic and/or predictive value of TIMP-1 analyses.

Jensen SB, Mouridsen HT, Bergmann OJ, Reibel J, Brünner N, Nauntofte B. · Department of Oral Medicine, Clinical Oral Physiology, Oral Pathology & Anatomy, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Denmark. · Oral Surg Oral Med Oral Pathol Oral Radiol Endod. · Pubmed #18554960 No free full text.

Abstract: OBJECTIVE: The aim of the study was to examine oral mucosal lesions, microbial changes, and taste disturbances induced by adjuvant chemotherapy (CT) in breast cancer patients during and 1 year after treatment. STUDY DESIGN: Forty-five consecutive breast cancer patients, eligible for adjuvant CT with cyclophosphamide, epirubicin or methotrexate, and 5-fluorouracil were followed before, during, 6 months and 1 year after CT and were compared to a control group of 31 breast cancer patients not receiving adjuvant CT. RESULTS: During CT, oral mucosal lesions developed including erythema (n = 10, 22%) and ulceration (n = 7, 16%). Five patients (11%) were diagnosed with oral candidosis. Scores of dental bacterial plaque and gingival inflammation increased during CT and the oral microbial composition changed towards a more acidophilic flora. Taste disturbances were experienced by 84% (n = 38) of the patients in the CT group. CONCLUSION: In breast cancer patients, moderate-intensive adjuvant CT caused oral mucosal lesions, oral candidosis, taste disturbances and a more acidophilic oral microflora. These adverse effects were temporary and the majority of the patients were mildly affected.

Offersen BV, Alsner J, Ege Olsen K, Riisbro R, Brünner N, Sørensen FB, Sørensen BS, Schlemmer BO, Overgaard J. · Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark. · Acta Oncol. · Pubmed #18465330 No free full text.

Abstract: BACKGROUND: The prognostic potential of HER2, TP53 mutations, PAI-1 protein levels, angiogenesis and proliferation were investigated in tumours from 408 patients with early breast cancer followed >10 years. One hundred and sixty seven patients (41%) died from breast cancer. MATERIALS AND METHODS: Tumour sections were stained for HER2, CD34, and MIB-1. HER2 scores were based on staining intensity, 3+ being considered HER2+. Angiogenesis was scored by the Chalkley method. MIB-1 was evaluated using systematic random sampling. PAI-1 was measured by ELISA. TP53 mutations were evaluated by DGGE analysis and DNA sequencing. RESULTS: Ninety one patients (22%) were HER2 positive. TP53 was mutated in 101 cases (25%). Median PAI-1, Chalkley and MIB-1 was 0.72 ng/mg protein (range, 0-90 ng/mg protein), 5.00 (range, 2.67-12.00) and 15% (range, 1-83%). MIB-1 was correlated with HER2+, Chalkley counts, TP53 mutations (all p <0.0001), and PAI-1 (p =0.002). In univariate analyses with DSS as endpoint, HER2+ (p <0.0001), mutated TP53 (p <0.0001), high Chalkley (p =0.008), MIB-1 (p =0.002), tumour size (p =0.008), grade (p <0.0001), negative estrogen receptor (p =0.0001), and lymph node status (p <0.0001) were prognostic markers. Among node-negative patients, HER2+ (p =0.0002), mutated TP53 (p =0.001), high PAI-1 levels (p =0.02), and grade (p =0.03) indicated poor DSS. In node-positive patients, HER2+ (p =0.0002), mutated TP53 (p <0.0001), MIB-1 (p =0.01), Chalkley scores (p =0.007), negative estrogen receptor (p <0.0001) and grade (p =0.001) indicated poor prognosis. In multivariate analysis, metastatic nodes (1-3 positive: RR 1.56 95% CI 1.02-2.38; >3 positive: RR 3.70 95% CI 2.54-5.38), HER2+ (RR 1.91, 95% CI 1.35-2.70), mutated TP53 (RR 1.70, 95% CI 1.21-2.38), PAI-1 (RR 1.04, 95% CI 1.01-1.07) and grade 3 (RR 1.96, 95% CI 1.83-3.22) were independent markers of poor outcome. CONCLUSION: Compared to PAI-1 protein levels, Chalkley counts and MIB-1, HER2+ and mutations of TP53 were the strongest independent markers of poor prognosis irrespective of nodal status.

Kuvaja P, Würtz SØ, Talvensaari-Mattila A, Brünner N, Pääkkö P, Turpeenniemi-Hujanen T. · Department of Oncology and Radiotherapy, Oulu University Hospital, University of Oulu, Oulu, Finland. · Cancer Biomark. · Pubmed #18048967 No free full text.

Abstract: A number of studies have demonstrated that high tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) are associated with a poor prognosis in breast cancer, suggesting that TIMP-1 could be a valid prognostic marker in this disease. Recently, our laboratories have presented results showing that TIMP-1 also carries prognostic information when measured in serum. This is an important finding, since serum is a much more preferable material compared with tumor tissue extracts. The aim of the present study was to validate the previous results concerning the prognostic value of TIMP-1 in serum obtained preoperatively from 68 patients with primary breast cancer. This was done by measuring the same serum samples as in the previous study but in a different laboratory using a different ELISA assay. We confirmed that patients with the highest serum levels of TIMP-1 (> 197.7 ng/ml) had significantly shorter disease-specific survival compared with patients with low serum TIMP-1 levels. In the group of node-negative patients, 53% of the patients with high levels of TIMP-1 survived after 10 years of follow-up compared to 92% of the patients with low levels. This study thus confirms the reproducibility across laboratories of the results concerning the prognostic value of TIMP-1 in serum. We also investigated whether measurements of the specific fraction of uncomplexed TIMP-1 improved the prognostic value of TIMP-1 in serum, as has been shown to be the case for tumor tissue extracts. However, including information of the level of uncomplexed TIMP-1 did not seem to provide additional prognostic information to that already provided by total TIMP-1.

Würtz SØ, Møller S, Mouridsen H, Hertel PB, Friis E, Brünner N. · Department of Veterinary Pathology, The Faculty of Life Sciences, University of Copenhagen, Ridebanevej 9, DK-1870 Frederiksberg C, Denmark. · Mol Cell Proteomics. · Pubmed #17998244 links to  free full text

Abstract: 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.

Offersen BV, Riisbro R, Knoop A, Brünner N, Overgaard J, Anonymous00056. · Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus C, Denmark. · Acta Oncol. · Pubmed #17653901 No free full text.

Abstract: Plasminogen Activator Inhibitor type-1 (PAI-1) is involved in tumor invasion and progression. High levels of PAI-1 are associated with poor prognosis in breast cancer, and PAI-1 has been shown to play a role in angiogenic processes. Since estimates of tumor angiogenesis may predict poor prognosis we studied the relationship between PAI-1 and estimates of angiogenesis in breast cancer. Tumor tissue specimens from 438 breast cancer patients were included. Median follow-up was 10.3 years. Protein levels of PAI-1 were measured using an ELISA. Angiogenesis scores were performed using a Chalkley grid. Median PAI-1 level was 0.70 ng/mg protein (range, 0 - 90 ng/mg protein) and median Chalkley count was 5.00 (range, 2.67 - 12.00). Chalkley counts were not correlated with PAI-1. In univariate analysis both increasing PAI-1 and increasing Chalkley counts evaluated as continuous parameters were significantly associated with poor disease-specific survival with RR 1.04 (95% CI 1.02 - 1.07) (p<0.0001) and RR 1.11 (95% CI 1.01 - 1.22) (p=0.04), respectively. High tertiles of PAI-1 were borderline significantly correlated with poor disease-specific survival (p=0.06), whereas high tertiles of Chalkley counts were significantly associated with poor disease-specific survival (p=0.004). Combining low/low versus high/high tertiles of Chalkley counts and PAI-1 showed actuarial 10-year survival rates of 82% versus 52% (p=0.004). High N-stage (p<0.0001), grade (p<0.0001) and increasing levels of PAI-1 (p=0.009) were independent markers of death from breast cancer. This study confirms high PAI-1 or high Chalkley counts as markers of poor prognosis in breast cancer patients, and suggests that the prognostic impact of PAI-1 is independent of its supposed involvement in tumor angiogenesis.

Jensen SB, Mouridsen HT, Reibel J, Brünner N, Nauntofte B. · Department of Oral Medicine, Clinical Oral Physiology, Oral Pathology and Anatomy, Institute of Odontology, Faculty of Health Sciences, University of Copenhagen, Norre Allé 20, DK-2200 Copenhagen N, Denmark. <> · Oral Oncol. · Pubmed #17588802 No free full text.

Abstract: It is an open question if chemotherapy (CT) per se imposes adverse effects on salivary gland function. The aim of the present study was to investigate effects of CT on salivary function in breast cancer patients during and after adjuvant CT. Forty-five breast cancer patients, eligible for adjuvant CT with CEF or CMF (cyclophosphamide, epirubicin or methotrexate, 5-fluorouracil) were followed before, during, six months and one year after CT. Findings were compared to those in a control group of 31 breast cancer patients not receiving CT. Flow rates and compositions of unstimulated and stimulated whole saliva as well as stimulated parotid saliva (UWS, SWS and SPS) were measured. Feeling of oral dryness and saliva-related complaints were registered. UWS and SWS flow rates decreased during CT (p<0.001 and p<0.01). UWS remained lower six months after, but reached baseline level within one year. SPS flow rate was not significantly affected, suggesting that the decrease in whole saliva production is accounted for by decreased acinar saliva formation by the submandibular glands. Twenty patients (44%) suffered from hyposalivation (UWS < or = 0.1 ml/min and/or SWS < or = 0.5 ml/min) during CT. Xerostomia scores rose during CT and stayed elevated one year after treatment. CT also induced compositional changes by slightly increasing salivary sodium and chloride concentrations as well as decreasing inorganic phosphate concentrations in spite of lower or unchanged flow rates, implying that ductal modification mechanisms are affected. UWS and SWS total protein output and UWS secretory IgA output decreased in response to CT. Thus, the results suggest that acinar and ductal cell functions are affected by adjuvant CT. These adverse drug reactions are temporary, as salivary findings generally returned to baseline values within one year following treatment.

Sieuwerts AM, Usher PA, Meijer-van Gelder ME, Timmermans M, Martens JW, Brünner N, Klijn JG, Offenberg H, Foekens JA. · Department of Medical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands. · Clin Chem. · Pubmed #17478562 links to  free full text

Abstract: BACKGROUND: TIMP-1 protein is a prognostic factor for recurrence-free and overall survival (OS) time in breast cancer. We evaluated the prognostic value of TIMP1 mRNA and a novel TIMP1 mRNA splice variant in 1301 primary breast cancer patients. METHODS: We measured mRNA transcripts of full-length TIMP1 (TIMP1-v1) and the novel splice variant lacking exon 2 (TIMP1-v2) by use of real-time RT-PCR in frozen primary tumor samples. Transcript concentrations are correlated with histomorphological and biological factors, TIMP-1 protein, and distant metastasis-free survival (MFS) and OS time. RESULTS: TIMP1-v1 and TIMP1-v2 alone were not informative with respect to predicting prognosis. However, the PCR assay designed to measure the combination of v1 + v2 showed that high concentrations of this combination were associated with good prognosis. In Cox multivariate regression analysis, which also included the traditional prognostic factors, increasing concentrations were independently associated with prolonged MFS (P = 0.004) and OS (P = 0.048). Including TIMP-1 protein and TIMP1-v1+v2 mRNA together in the multivariate model revealed that protein and mRNA were both independently associated with prognosis, with hazard ratios pointing in opposite directions. CONCLUSION: High concentrations of TIMP1-v1+2 mRNA are associated with good prognosis in patients with primary breast cancer. Since high concentrations of TIMP-1 protein are associated with poor prognosis, the presence of possible posttranscriptional mechanisms requires further investigation.

Schou M, Brünner N, Spang-Thomsen M, Rygaard J. · Bartholin Instituttet, Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark. · APMIS. · Pubmed #17207091 No free full text.

Abstract: Most nude mice do not allow the formation of metastases after heterotransplantation of human malignant tumours. Here we describe a substrain of BALB/c nude mice (BALB/c/AnNCr) that reproducibly allows some human cancers to metastasize. By Mendelian analysis of hybrids between this substrain and C57BL/6J +/+ mice we found that the ability to allow a human tumour (MDA-MB-435 BAG) to express its metastatic phenotype is determined by a recessively inheritable trait in the mouse host. We are presently working to identify the genetics responsible for development of metastases. The study also includes immunohistochemical and electron microscopic analysis of the test tumour, originally assumed to be a human mammary carcinoma, but shown to possess characteristics of a malignant melanoma (1). The ultimate aim of our ongoing study is to establish a substrain of nude mice that will allow metastasis in all recipients.

Schrohl AS, Meijer-van Gelder ME, Holten-Andersen MN, Christensen IJ, Look MP, Mouridsen HT, Brünner N, Foekens JA. · Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark. · Clin Cancer Res. · Pubmed #17114213 links to  free full text

Abstract: PURPOSE: Only about 50% of metastatic breast cancer patients benefit from cytotoxic chemotherapy. Today, no validated markers exist for prediction of chemotherapy sensitivity/resistance in this patient group. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to protect against apoptosis, and the purpose of the present study was to test the hypothesis that tumors expressing high levels of TIMP-1 are protected against apoptosis-inducing agents and thus less sensitive to apoptosis-inducing chemotherapeutic drugs. EXPERIMENTAL DESIGN: We investigated the association between primary tumor expression levels of TIMP-1 protein and objective response to first-line chemotherapy in 173 patients with metastatic breast cancer. RESULTS: When analyzed as a continuous log-transformed variable, increasing TIMP-1 levels were significantly associated with lack of response to cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemotherapy (P = 0.01; odds ratio, 2.0; 95% confidence interval, 1.1-3.3). In a multivariate model, including lymph node status, steroid hormone receptor status, menopausal status, dominant metastases site, type of chemotherapy, and disease-free interval, TIMP-1 was significantly associated with resistance to treatment (P = 0.03; odds ratio, 1.7; 95% confidence interval, 1.1-3.3). CONCLUSIONS: In the present exploratory study, we showed that elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to chemotherapy. By using TIMP-1, we identified a group of patients with metastatic breast cancer, which hardly respond to the most frequently used chemotherapy regimes (i.e., cyclophosphamide/methotrexate/5-fluorouracil and anthracyclines).

Pappot H, Pedersen AN, Brünner N, Christensen IJ. · The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark. · Lung Cancer. · Pubmed #16325301 No free full text.

Abstract: In a lung cancer population comprising tumor tissue from 99 pulmonary adenocarcinoma patients, the relationship between tumor tissue level of the complex formed of urokinase (uPA) and its type-1 inhibitor (PAI-1) and survival was studied. The study included patient material previously investigated for the prognostic impact of PAI-1 on survival. Standard clinical parameters were available and the patients had a median survival time of 25 months. An ELISA established to measure preformed uPA-PAI-1 complexes was applied to the tumor extracts and previously measured data on uPA and PAI-1 levels were available. The amounts of uPA-PAI-1 complex measured in pulmonary adenocarcinoma tissue were within the same range as previously reported in breast cancer tissue (0.11-5.74 ng/mg protein). uPA and PAI-1 levels were weakly correlated to the uPA-PAI-1 complex, r = 0.52 and r = 0.47, respectively, and no relation was found between uPA-PAI-1 complex and any of the clinical parameters. However, a significant prognostic impact of PAI-1 on prognosis was demonstrated (HR = 1.62, p = 0.04). Patients with high PAI-1 and low uPA-PAI-1 complex were found to have a significantly poorer survival than patients with low PAI-1 and high uPA-PAI-1 complex (HR = 3.06, p = 0.01). This is the first investigation of the prognostic impact of uPA-PAI-1 complex in a tumor type other than breast cancer, showing low levels of uPA-PAI-1 complex in combination with high levels of PAI-1 to be associated with poor prognosis. To understand these interactions and the clinical importance of the tissue levels of uPA, PAI-1 and uPA-PAI-1 complex, the results suggest further exploratory studies of the components in pulmonary adenocarcinomas and other cancers.

Grebenchtchikov N, Maguire TM, Riisbro R, Geurts-Moespot A, O'Donovan N, Schmitt M, McGreal G, McDermott E, O'Higgins N, Brünner N, Sweep CG, Duffy MJ. · Department of Chemical Endocrinology, University Medical Centre Nijmegen, Nijmegen, The Netherlands. · Oncol Rep. · Pubmed #15944795 No free full text.

Abstract: The plasminogen activator (PA) system comprises the 2 serine proteases, urokinase PA (uPA) and tissue PA (tPA), the 2 serpin inhibitors, PAI-1 and PAI-2 and the uPA receptor (uPAR; CD87). High levels of uPA, PAI-1, uPA-PAI-1 complex and uPAR in breast cancer tissue are associated with poor prognosis, while high levels of tPA or PAI-2 correlate with good prognosis. In this study, pre-operative plasma levels of uPA, PAI-1, uPAR, tPA, uPA-PAI-1 complex, and tPA-PAI-1 complex were measured in patients with benign (n=103) and malignant breast disease (n=113) by immunoenzymatic assays (ELISA). While plasma antigen levels of uPA, PAI-1, uPA-PAI-1 complex and uPAR were not significantly different in the 2 groups, antigen levels of tPA and tPA-PAI-1 complex were significantly higher in patients with breast carcinoma compared to the control group. In plasma from the breast cancer patients, uPA levels correlated weakly but significantly with those of tPA (r=0.20, p=0.035) and uPAR (r=0.208, p=0.028). tPA levels correlated strongly with tPA-PAI-1 complex (r=0.972, p=0.0001) while uPA-PAI-1 levels were significantly associated with PAI-1 levels (r=0.534, p<0.0001), tPA levels (r=0.348, p=0.0003) and tPA-PAI-1 levels (r=0.356, p=0.002). However, no significant correlation was found between plasma and tumor tissue levels of uPA, PAI-1, uPA-PAI-1 complex, tPA or tPA-PAI-1. Our findings indicate that determination of these factors in plasma do not reflect their concentration in tumor tissue. Therefore, measurement of PA components in blood cannot be recommended for assessing prognosis in breast cancer.

Larsen MB, Stephens RW, Brünner N, Nielsen HJ, Engelholm LH, Christensen IJ, Stetler-Stevenson WG, Høyer-Hansen G. · The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark. · Scand J Immunol. · Pubmed #15882437 No free full text.

Abstract: Tissue inhibitor of metalloproteinases (TIMP)-2 is a highly conserved molecule, which binds both active and latent matrix metalloproteinase (MMP)-2. TIMP-2 is also involved in the activation of MMP-2 on the cell surface. A quantitative enzyme-linked immunosorbent assay (ELISA) was established and optimized for measurement of TIMP-2 in plasma. The capturing antibody in the ELISA was a monoclonal, while the detecting antibody was a chicken polyclonal antibody recognizing the native form of human TIMP-2. The levels of TIMP-2 were measured in ethylenediaminetetraacetic acid (EDTA) and citrate plasma from healthy donors. The median values were determined as 163 ng/ml (n = 186) with a range of 109-253 ng/ml for EDTA plasma and 139 ng/ml (n = 77) with a range of 95-223 ng/ml for citrate plasma. The TIMP-2 concentration in citrate plasma from 15 patients with advanced, stage IV breast cancer had a median value of 160 ng/ml, only slightly higher but statistically distinguishable from the level found in citrate plasma from the healthy donors. In addition, the TIMP-2 concentration in EDTA plasma from colorectal cancer patients revealed a significantly higher level in plasma from patients with Dukes stage A (P = 0.01) compared with patients with more advanced Dukes stages.

Würtz SØ, Christensen IJ, Schrohl AS, Mouridsen H, Lademann U, Jensen V, Brünner N. · Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Ridebanevej 9, DK-1870 Frederiksberg C. · Mol Cell Proteomics. · Pubmed #15640527 links to  free full text

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.

Meijer-van Gelder ME, Look MP, Peters HA, Schmitt M, Brünner N, Harbeck N, Klijn JG, Foekens JA. · Department of Medical Oncology, Erasmus-MC Rotterdam, Rotterdam, The Netherlands. · Cancer Res. · Pubmed #15231667 links to  free full text

Abstract: The prognostic value of components of the urokinase-type plasminogen activator (uPA) system, its receptor uPAR (CD87), and plasminogen activator inhibitors PAI-1 and PAI-2 is well established. We studied the predictive value of these proteolytic factors by evaluating the association of their tumor expression level and the efficacy of tamoxifen therapy in patients with recurrent breast cancer. The antigen levels of the four factors were determined by ELISA in cytosols prepared from estrogen receptor-positive primary breast tumors of 691 hormone-naive breast cancer patients with recurrent disease and treated with tamoxifen as first-line systemic therapy. High tumor levels of uPA (P < 0.001), uPAR (P < 0.01), and PAI-1 (P = 0.01) were associated with a lower efficacy of tamoxifen therapy. In the multivariable analysis, uPA (P < 0.001) provided additional information independent of the traditional predictive factors to predict benefit from tamoxifen therapy. High levels of uPA, uPAR, and PAI-1 predicted a shorter progression-free survival (PFS) on tamoxifen in an analysis of the first 9 months of therapy. However in the analysis during the total follow-up period, high PAI-2 levels (P = 0.01) showed a longer response to tamoxifen. In conclusion, uPA, uPAR, and PAI-1, components of the urokinase system, are predictive for the efficacy of tamoxifen therapy in patients treated for recurrent breast cancer. Knowledge of their tumor expression levels might be helpful for future individualized therapy protocols, including possible new-targeted therapies based on the interference in the urokinase system.

Schrohl AS, Holten-Andersen MN, Peters HA, Look MP, Meijer-van Gelder ME, Klijn JG, Brünner N, Foekens JA. · The Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Frederiksberg C, Denmark. · Clin Cancer Res. · Pubmed #15073104 links to  free full text

Abstract: PURPOSE: In the present study, we investigated the association between tumor tissue levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prognosis in patients with primary breast cancer and analyzed whether TIMP-1 may be useful as a prognostic marker in combination with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1). EXPERIMENTAL DESIGN: In cytosolic extracts of 2984 primary breast tumors, total levels of TIMP-1 were determined using an established, validated ELISA. Levels of uPA and PAI-1 have previously been determined in the extracts. RESULTS: Univariate survival analysis showed a significant relationship between higher levels of TIMP-1 (continuous log-transformed variable) and poor prognosis [recurrence-free survival (RFS), overall survival (OS); P < 0.001]. Performing isotonic regression analysis, we identified a cut point to classify tumors as TIMP-1-low or TIMP-1-high. Using this cut point, high levels of TIMP-1 were significantly associated with shorter survival in univariate analysis, both in the total patient group (RFS, OS; P < 0.001), in the node-negative subgroup (RFS, hazard ratio = 1.28, P = 0.006), and in the node-positive subgroup (RFS, hazard ratio = 1.43, P < 0.001). In multivariate analysis, including uPA and PAI-1, TIMP-1 was significantly associated with shorter RFS, both when included as a continuous log-transformed (P = 0.03) and as a dichotomized variable (P = 0.002). CONCLUSIONS: This study validates previous findings that tumor tissue levels of TIMP-1 are associated with prognosis in patients with primary breast cancer. It confirms that TIMP-1 may be useful as a prognostic marker in combination with uPA/PAI-1 and adds substantial positive information on the use of TIMP-1 as a prognostic marker in breast cancer.

Amhlaoibh RN, Hoegh-Andersen P, Brünner N, Sørensen A, Winding B, Holst-Hansen C, Karsdal MA, Engsig MT, Delaissé JM, Heegaard AM. · Nordic Bioscience A/S, Herlev, Denmark. · Clin Exp Metastasis. · Pubmed #15065604 No free full text.

Abstract: The Arguello model of cancer metastasis to bone has been used extensively to study breast cancer-induced osteolytic disease. The effects of therapy on skeletal disease and on tumour burden in soft organs are traditionally measured using radiography and/or time-consuming histomorphometry, respectively. The purpose of this study was to develop a sensitive and efficient method for evaluating tumour burden in vivo using MDA-231 cells transduced with the E. coli lacZ gene (MDA-231BAG). Osteolysis was measured by radiography and tumour burden was measured histomorphometrically or biochemically. In untreated mice, measurements of tumour burden in bone extracts using human cytokeratin-associated tissue polypeptide antigen (TPA) ELISA or E. coli beta-galactosidase (beta-gal) activity immunoassay reflected the extent of osteolytic disease as measured by radiography; however, tumour load could be detected before onset of osteolysis. When monitoring the effect of therapy (0.2 mg/kg ibandronate/day), radiography alone proved to be insufficient. Mice treated with the bisphosphonate ibandronate from time of inoculation with cancer cells had no radiologically visible signs of osteolysis but significant tumour load was measured in the bone extracts using these assays. Furthermore, beta-gal activity could be used as a measurement of tumour load in soft organs, and unlike other human breast cancer markers expressed by the MDA-231 cells in vitro, beta-gal activity was detected in the serum of mice with progressive disease. In conclusion, we describe an efficient model of breast cancer-induced osteolysis to quantify the effect of therapy on disease load and distribution, which could be beneficial in evaluating novel therapies for the treatment of the disease.

Ye JJ, Liang SJ, Guo N, Li SL, Wu AM, Giannini S, Sachdev D, Yee D, Brünner N, Ikle D, Fujita-Yamaguchi Y. · Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. · Horm Metab Res. · Pubmed #14710366 No free full text.

Abstract: Proliferative and anti-apoptotic actions of IGFs are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and -II bind with high affinity. We previously reported that alphaIGF-IR scFv-Fc (scFv-Fc) consisting of the alphaIGF-IR scFv and human IgG (1) Fc domain retained general characteristics of the parental 1H7 monoclonal antibody, and significantly suppressed MCF-7 tumor growth. We proposed IGF-IR down-regulation as a possible mechanism for inhibition of MCF-7 tumor growth. To further determine the therapeutic potentials of this approach, in vivo effects of this antibody on breast tumor growth were evaluated in the absence or presence of tamoxifen (Tam) using a T61 human breast tumor model. T61 xenograft growth in athymic mice was compared under five conditions, PBS, scFv-Fc, Tam, scFv-Fc+Tam, and control antibody. While treatment with PBS and control antibody did not affect T61 tumor growth, scFv-Fc, Tam, and scFv-Fc+Tam treatments significantly suppressed the tumor growth during the first two weeks of treatment. Although the growth inhibitory effect of scFv-Fc during the first two weeks was significant, the tumor grew as rapidly as PBS-treated tumors thereafter. This rapid tumor growth was suppressed when scFv-Fc was combined with Tam. Throughout four weeks, the combined Tam+scFv-Fc treatment was more effective in inhibiting the T61 tumor growth than scFv-Fc or Tam treatment alone. scFv-Fc treatment down-regulated IGF-IR which appears to contribute to tumor growth inhibition. This study provides evidence that simultaneous targeting of IGF-IR and the estrogen receptor may enhance the therapeutic effect.

Hemsen A, Riethdorf L, Brünner N, Berger J, Ebel S, Thomssen C, Jänicke F, Pantel K. · Institute for Tumor Biology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20251 Hamburg, Germany. · Int J Cancer. · Pubmed #14601049 No free full text.

Abstract: The urokinase-type plasminogen activator receptor (uPAR, CD87) plays a central role in the plasminogen activation cascade, which participates in extracellular matrix degradation, cell migration and invasion. Here we performed a comprehensive immmunohistochemical evaluation of uPAR expression in primary tumor cells, tumor-surrounding fibroblasts, lymph node metastases and micrometastatic cells in bone marrow of patients with breast carcinomas at the time of primary diagnosis. Variable degrees of uPAR staining of tumor cells were observed in 84 of 93 (90%) carcinomas, whereas intratumoral fibroblasts were uPAR-positive in 70 (75%) carcinomas. The fraction of uPAR-positive primary tumor cells but not fibroblasts was positively correlated with the presence of tumor cells in bone marrow (p = 0.037), whereas no correlation with lymph node metastasis was found. Immunophenotyping of bone marrow and lymph node specimens revealed expression of uPAR on metastatic tumor cells in 10 of 13 and 22 of 23 cases, respectively. Direct comparison to the autologous primary tumor cells showed different uPAR staining scores in most patients with evidence for both up- and downregulation of uPAR on metastatic cells. Our results indicate that uPAR plays an active role in breast cancer metastasis and may therefore be a promising target for new biologic therapies.

Look M, van Putten W, Duffy M, Harbeck N, Christensen IJ, Thomssen C, Kates R, Spyratos F, Fernö M, Eppenberger-Castori S, Fred Sweep CG, Ulm K, Peyrat JP, Martin PM, Magdelenat H, Brünner N, Duggan C, Lisboa BW, Bendahl PO, Quillien V, Daver A, Ricolleau G, Meijer-van Gelder M, Manders P, Edward Fiets W, Blankenstein M, Broët P, Romain S, Daxenbichler G, Windbichler G, Cufer T, Borstnar S, Kueng W, Beex L, Klijn J, O'Higgins N, Eppenberger U, Jänicke F, Schmitt M, Foekens J, Bendah PO. · Erasmus MC, Josephine Nefkens Institute, Rm. Be 428, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. · Thromb Haemost. · Pubmed #12958624 No free full text.

Abstract: In this report we present an extension of the pooled analysis of the prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I in breast cancer patients. We analyzed a different endpoint, metastasis-free survival (MFS). We checked the consistency of the estimates for uPA and PAI-1 for relapse-free survival (RFS) and MFS exploring possible sources of heterogeneity. Nodal status, the most important prognostic factor for breast cancer, introduced heterogeneity in the uPA/PAI-1 survival analyses, reflecting the interaction between nodal status and uPA/PAI-1. The estimates for uPA and PAI-1 were found to be consistent, even when a different transformation of their values was used. The heterogeneity of the separate data sets decreased if the levels of uPA and PAI-1 were ranked, data sets were pooled, and the analyses corrected for the base model that included all traditional prognostic factors, and stratified by data set. We conclude that uPA and PAI-1 are ready to be used in the clinic to help classify breast cancer patients into high and low risk groups.