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Erythroferrone (ERFE), the erythroid regulator of iron metabolism, inhibits hepcidin to increase iron availability for erythropoiesis. ERFE plays a pathological role during ineffective erythropoiesis as occurs in X-linked sideroblastic anemia (XLSA) and β-thalassemia. Its measurement might serve as an indicator of severity for these diseases. However, for reliable quantification of ERFE analytical characterization is indispensable to determine the assay's limitations and define proper methodology. We developed a sandwich ELISA for human serum ERFE using polyclonal antibodies and report its extensive analytical validation. This new assay showed, for the first time, the differentiation of XLSA and β-thalassemia major patients from healthy controls (p = 0.03) and from each other (p<0.01), showing the assay provides biological plausible results. Despite poor dilution linearity, parallelism and recovery in patient serum matrix, which indicated presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte, our assay correlated well with two other existing ERFE ELISAs (both R2 = 0.83). Nevertheless, employment of one optimal dilution of all serum samples is warranted to obtain reliable results. When adequately performed, the assay can be used to further unravel the human erythropoiesis-hepcidin-iron axis in various disorders and assess the added diagnostic value of ERFE., Competing Interests: The authors have declared that no competing interests exist.

In a previous study, we detected a significant association between phosphoserine aminotransferase 1 (PSAT1) hyper-methylation and mRNA levels to outcome to tamoxifen treatment in recurrent disease. We here aimed to study the association of PSAT1 protein levels to outcome upon tamoxifen treatment and to obtain more insight in its role in tamoxifen resistance. A cohort of ER positive, hormonal therapy naïve primary breast carcinomas was immunohistochemically (IHC) stained for PSAT1. Staining was analyzed for association with patient's time to progression (TTP) and overall response on first-line tamoxifen for recurrent disease. PSAT1 mRNA levels were also assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR; n = 161) and Affymetrix GeneChip (n = 155). Association of PSAT1 to biological pathways on tamoxifen outcome were assessed by global test. PSAT1 protein and mRNA levels were significantly associated to poor outcome to tamoxifen treatment. When comparing PSAT1 protein and mRNA levels, IHC and RT-qPCR data showed a significant association. Global test results showed that cytokine and JAK-STAT signaling were associated to PSAT1 expression. We hereby report that PSAT1 protein and mRNA levels measured in ER positive primary tumors are associated with poor clinical outcome to tamoxifen.

Background: The use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are subject to a variety of interferences., Methods: In the present study, the authors have tested the ELISA methods for quantifying bevacizumab (BVZ) to investigate this interference. Three different ELISA methods were used and exhibited similar characteristics., Results: The detection limits of the ELISA methods varied from 0.05 to 0.07 ng/mL. To monitor assay performance, BVZ was measured in a control sample during each run. The BVZ concentration in the control sample was 15.4 μg/mL, the within-run imprecision (CV) and between-run CV were 4.3% and 10.4% (direct ELISA), 5.2% and 12.9% (indirect/Rabbit ELISA), and 3.9% and 9.1% (indirect/Chicken ELISA). The assays exhibited good precision and parallelism in serial dilutions of samples and a mean recovery of 98% (range, 78%-118%)., Conclusions: The authors show that the degree of interference by using direct and indirect target immobilization depends heavily on the method of target immobilization on the surface of the ELISA plate, and is patient-specific. The results highlight pitfalls of potential relevance to sandwich-type assays, and an approach to rectify such problems. This approach will yield a valid assay protocol for the measurement of monoclonal therapeutic antibodies in case no target is available for direct immobilization.

Background: Tribbles homolog 3 (TRIB3) is a pseudokinase involved in the regulation of several signaling pathways involved in cell survival and/or cell stress. Here, we determined the correlation between breast cancer prognosis and TRIB3 protein levels and established the role of TRIB3 in cell survival after hypoxia and/or radiotherapy., Material and Methods: TRIB3 mRNA and protein were quantified in a new independent breast cancer patient cohort using QPCR and a new specific avian antibody against TRIB3. In addition, we used siRNA-mediated knockdown of TRIB3 in a colony-forming assay after hypoxia and radiotherapy., Results: TRIB3 mRNA and protein levels did not correlate in breast cancer cell lines or human breast cancer material. We validated our earlier finding that high TRIB3 mRNA denotes a poor prognosis, but found that high TRIB3 protein levels were associated with a good prognosis in breast cancer patients. We also show that knockdown of TRIB3 resulted in an increased survival under hypoxic conditions., Conclusion: Whereas mRNA levels of TRIB3 are related with a poor prognosis, TRIB3 protein is associated with a good prognosis in human breast cancer patients, possibly due to the fact that TRIB3 is involved in hypoxia tolerance., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Hepcidin is an iron-regulatory peptide hormone that consists of 3 isoforms: bioactive hepcidin-25, and inactive hepcidin-22 and hepcidin-20. Hepcidin is instrumental in the diagnosis and monitoring of iron metabolism disorders, but reliable methods for its quantification in serum are sparse, as is knowledge of their relative analytical strengths and clinical utility., Methods: We developed a competitive (c)-ELISA and an immunocapture TOF mass-spectrometry (IC-TOF-MS) assay. Exploiting these 2 methods and our previously described weak cation exchange (WCX)-TOF-MS assay, we measured serum hepcidin concentrations in 186 patients with various disorders of iron metabolism and in 23 healthy controls., Results: We found that (a) the relative differences in median hepcidin concentrations in various diseases to be similar, although the absolute concentrations measured with c-ELISA and WCX-TOF-MS differed; (b) hepcidin isoforms contributed to differences in hepcidin concentrations between methods, which were most prominent in patients with chronic kidney disease; and (c) hepcidin concentrations measured by both the c-ELISA and IC-TOF-MS correlated with ferritin concentrations <60 μg/L, and were suitable for distinguishing between iron deficiency anemia (IDA) and the combination of IDA and anemia of chronic disease., Conclusions: c-ELISA is the method of choice for the large-scale quantification of serum hepcidin concentrations, because of its low limit of detection, low cost, and high-throughput. Because of its specificity for bioactive hepcidin-25, WCX-TOF-MS can be regarded as a valuable special-purpose assay for disorders with variable concentrations of hepcidin isoforms, such as chronic kidney disease.

Pathogenic bacteria, including Pseudomonas aeruginosa, interact with and engage the host plasminogen (Plg) activation system, which encompasses the urokinase (uPA)-type Plg activator, and is involved in extracellular proteolysis, including matrilysis and fibrinolysis. We hypothesized that secreted bacterial proteases might contribute to the activation of this major extracellular proteolytic system, thereby participating in bacterial dissemination. We report that LasB, a thermolysin-like metalloprotease secreted by Ps. aeruginosa, converts the human uPA zymogen into its active form (kcat=4.9 s-1, Km=8.9 microM). Accordingly, whereas the extracellular secretome from the LasB-expressing pseudomonal strain PAO1 efficiently activates pro-uPA, the secretome from the isogenic LasB-deficient strain PDO240 is markedly less potent in pro-uPA activation. Still, both secretomes induce some metalloprotease-independent activation of the human zymogen. The latter involves a serine protease, which we identified via both recombinant protein expression in Escherichia coli and purification from pseudomonal cultures as protease IV (PIV; kcat=0.73 s-1, Km=6.2 microM). In contrast, neither secretomes nor the pure proteases activate Plg. Along with this, LasB converts Plg into mini-Plg and angiostatin, whereas, as reported previously, it processes the uPA receptor, inactivates the plasminogen activator inhibitor 1, and activates pro-matrix metalloproteinase 2. PIV does not target these factors at all. To conclude, LasB and PIV, although belonging to different protease families and displaying quite different substrate specificities, both activate the urokinase-type precursor of the Plg activation cascade. Direct pro-uPA activation, as also reported for other bacterial proteases, might be a frequent phenomenon that contributes to bacterial virulence.

Certain serine proteases are considered to be signaling molecules that act through protease-activated receptors (PARs). Our recent studies have implicated PAR1 and PAR4 (thrombin receptors) and PAR2 (trypsin receptor) in human colon cancer growth. Here we analyzed the expression of KLK4, a member of the kallikrein-related peptidase (KLK) family of serine proteases and explored whether this member can activate PAR1 and PAR2 in human colon cancer cells. Immunohistochemistry showed KLK4 expression in human colon adenocarcinomas and its absence in normal epithelia. KLK4 (1 micromol/L) initiated loss of PAR1 and PAR2 from the HT29 cell surface as well as increased intracellular calcium transients in HT29 cells. This KLK4-induced Ca2+ flux was abrogated after an initial challenge of the cells with TRAP (SFLLR-NH2; 100 micromol/L), which is known to desensitize PAR1 and PAR2. Interestingly, PAR1 blocking antibody, which inhibits cleavage and activation by thrombin, dramatically reduced KLK4-induced Ca2+ influx, but blocking cleavage of PAR2 failed to attenuate the KLK4-induced Ca2+ flux. Consistently, desensitization with AP1 (TFFLR-NH2), targeting PAR1, attenuated most of the Ca2+ flux induced by KLK4. KLK4 also induced a rapid and significant ERK1/2 phosphorylation in HT29 cells. Our results demonstrate, for the first time, that KLK4 is aberrantly expressed in colon cancer and capable of inducing PAR1 signaling in cancer cells. These data suggest that KLK4 signaling via PAR1 may represent a novel pathway in colon tumorigenesis.

The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Despite its promise as a biomarker, the availability of high-sensitive hepcidin assays is still limited. We developed and validated a RadioImmunoAssay (RIA) to measure hepcidin quantitatively in human serum. This assay exhibited a very low detection limit (0.02 microg/l), low imprecision (coefficient of variation-range 4.4-6.2%) and good linearity and recovery (range: 81-105%). Hepcidin levels of samples of controls and patients with iron deficiency and inflammation showed an excellent correlation with our previously described quantitative time-of-flight mass spectrometry assay (range 2.5-266.8 microg/l, r = 0.92, P < 0.0001). The RIA detected: (i) differences in mean hepcidin levels between men (n = 29) and women (n = 35), (ii) differences between individuals of different HFE-genotypes (n = 60) and (iii) daily increases in hepcidin levels (n = 64). The assay (i) is easy to perform and many samples can be processed within one assay-run, (ii) shows accurate, reproducible and high-sensitive measurements and (iii) is anticipated to be particularly useful to study the effects of pathological and physiological stimuli on hepcidin levels in the lower range.

Objective: To analyse and compare the concentration of plasminogen activator (PA), urokinase-type PA (uPA), tissue-type PA (tPA), PA inhibitor (PAI)-1 and PAI-2, and the complexes uPA-PAI-1 and tPA-PAI-1 and calculated uPA and tPA uncomplexed with PAI-1 ('free') in urothelial cell carcinoma and matched benign urothelium, and in renal cell carcinoma (RCC) and matched benign renal tissue., Patients and Methods: Tissue samples were obtained during cystectomy (33 patients) and nephrectomy (55), and specific enzyme-linked immunosorbent assays were used to assess the PA components in extracts of these tissues., Results: Tissue levels of uPA-PAI-1 and tPA-PAI-1, but also PAI-1 itself, were greater in tumorous bladder and kidney tissue than in matched normal tissue (by 1.5-7.8 times). Free tPA was clearly lower in tumour tissue (by 0-0.12-fold). In bladder cancer, but not in RCC, levels of uPA (15.8-fold) and free uPA (16.4-fold) were greater in tumour tissue. Free uPA levels were less in RCC (0.41-fold). For both normal bladder and kidney tissue, there was no clear correlation between uPA-PAI-1 complex and either component. However, the formation of tPA-PAI-1 complexes in normal bladder and kidney tissue was primarily determined by PAI-1. Interestingly, in tumour tissues there was a strong, significant correlation between complex levels and both components., Conclusion: RCC and bladder cancer show distinct profiles of components of the PA system. This study provides a basis for further studies into both the (patho)physiological role of the PA system in these tumours, and into a possible relation with tumour progression and prognosis, and as target for therapy.

Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-alpha) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is sensitive, reliable and not susceptible to disturbances by interfering substances such as heterophilic antibodies. The assay involves a combination of four polyclonal antibodies. The antibodies, which capture the analyte, were raised in chicken and the trapping anti-analyte antibodies were raised in rabbit. The immobilization of capture antibodies was achieved via a coating antibody raised in a duck against chicken IgY and the recognition of trapping antibodies was achieved by a detection antibody raised in a goat against rabbit IgG and labelled with HRP. The analytical and functional sensitivities of the ELISA are 8 pg/mL and 13 pg/mL, respectively. The assay showed good precision and, in contrast to our in-house RIA, excellent parallelism in serial dilutions. The recovery of TNF-alpha spiked to plasma samples ranged from 97% to 119%. Comparison of the newly developed, sensitive ELISA with our in-house RIA showed that the median TNF-alpha value obtained by RIA (range: 0.095-10.0, median 0.578 ng/mL) was found to be 1.5-2 times higher than that obtained with the ELISA (range 0.008-5.84, median 0.213 ng/mL). Spearman correlation was 0.755 (p < 0.0001). In addition, analysis of the TNF-alpha concentrations in blood from healthy individuals and from patients suffering from tuberculosis, with RIA and ELISA, showed the same differences although TNF-alpha levels obtained with ELISA were lower. We feel that this ELISA is a major improvement compared to the currently available assays for TNF.

Purpose: BCAR1, the human homologue of the rat p130Cas protein, was identified in a functional screen for human breast cancer cell proliferation resistant to antiestrogen drugs. Here, we study the prognostic value of quantitative BCAR1 levels in a large series of breast cancer specimens., Experimental Design: A specific ELISA was developed to measure BCAR1 protein levels in 2593 primary breast tumor cytosols. Tumor levels of BCAR1 were correlated with relapse-free survival (RFS) and overall survival (OS) and compared with collected data on urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1)., Results: In tumor cytosols, BCAR1 protein levels varied between 0.02 and 23 ng/mg protein. BCAR1 levels exhibited a positive correlation with steroid hormone receptor levels, age and menopausal status, and uPA and PAI-1 levels. The level of BCAR1 (continuous or categorized as low, intermediate, or high) was inversely related with RFS and OS time. Multivariate analysis showed that BCAR1 levels contributed independently to a base model containing the traditional prognostic factors for both RFS and OS (both P < 0.0001). When added together with uPA and PAI-1 in the multivariate model, BCAR1 contributed independently of PAI-1 and was favored over uPA. Interaction tests allowed for additional analyses of BCAR1 protein levels in clinically relevant subgroups stratified by nodal and menopausal status., Conclusions: The quantitative BCAR1 protein level represents a prognostic factor for RFS and OS in primary breast cancer, independent of the traditional prognostic factors and the other novel marker PAI-1.

Background: High concentrations of breast cancer anti-estrogen resistance 1 (BCAR1) protein measured by Western blotting in primary breast tumor cytosols are associated with early disease progression and failure of tamoxifen therapy. The aim of the present study was to develop an ELISA to measure BCAR1 quantitatively in extracts of human breast cancer tissue., Methods: A recombinant fragment of BCAR1 (the human homolog of murine p130Cas) was produced in bacterial M15 cells, purified, and injected into chickens and rabbits. The generated antibodies were affinity-purified and used for the construction of an ELISA. After validation, the results obtained with the ELISA were compared with Western blot findings on primary breast tumors., Results: The detection limit the BCAR1 ELISA was 0.0031 microg/L, and the within-run imprecision (CV) was <20% at concentrations down to 0.004 microg/L. The within-run imprecision (CV) was 1.0-7.2%, and the between-run CV was 3.6-5.4%. There was no cross-reactivity with family member HEF1. The assay exhibited parallelism of results between serial dilutions and a mean recovery (range) of 96 (79-118)%., Conclusions: The ELISA measures BCAR1 in human breast cancer cytosols with high sensitivity and specificity. The assay can be used to confirm and to quantitatively extend previous semiquantitative Western blot data on the prognostic and predictive value of BCAR1 in human breast cancer; it can also be applied for other diseases.

Background: The ability of a solid tumor to grow and metastasize has a significant dependence on protease systems, such as the plasminogen activation system. The plasminogen activation system includes the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1), among other molecules. Both uPA and PAI-1 are established prognostic factors for patients with breast carcinoma. In the current study, the authors investigated whether the complex of uPA with PAI-1 is also associated with the natural course of this malignancy., Methods: Cytosolic levels of uPA, PAI-1, and the uPA:PAI-1 complex were measured in tumor tissue from 576 patients with lymph node-negative invasive breast carcinoma using quantitative enzyme-linked immunosorbent assays. Patients did not receive adjuvant systemic therapy, and the median follow-up duration was 61 months (range, 2-187 months) after primary diagnosis. Correlations with well known clinicopathologic factors were assessed, and univariate and multivariate survival analyses were performed., Results: uPA:PAI-1 complex levels were positively associated with adverse histologic grade and inversely correlated with estrogen and progesterone receptor status. On univariate analysis, increased levels of the uPA:PAI-1 complex were found to be associated with reduced recurrence-free survival (RFS) and overall survival (OS) rates. On multivariate analysis, uPA:PAI-1 complex levels were found to be an independent predictor of OS (P = 0.039), but not RFS (P = 0.240). When uPA and PAI-1 levels were not included in the multivariate analysis, uPA:PAI-1 complex levels became a significant predictor of both RFS and OS (P = 0.029 and P = 0.007, respectively)., Conclusions: The results of the current study demonstrate that uPA:PAI-1 complex levels have prognostic value on univariate analysis. In addition, increased uPA:PAI-1 complex levels were significantly associated with poor OS on multivariate analysis. Increased uPA:PAI-1 complex levels were also significantly associated with reduced RFS rates after the exclusion of uPA and PAI-1 levels from the multivariate analysis model.

It has been shown that urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-I) have predictive value for therapy success in advanced breast cancer. Levels of the complex between uPA and PAI-I, formed when both molecules are in their active form, might have superior predictive power. Here, we investigate the association between levels of uPA:PAI-I complex and rate of response to first-line systemic therapy for advanced breast cancer. Tumor tissues of 170 patients with advanced breast cancer were analyzed for uPA:PAI-I complex concentrations using a quantitative enzyme-linked immunosorbent assay. The patients received either endocrine therapy (n=96) or chemotherapy (n=74) as first-line treatment after diagnosis of advanced disease. Of the endocrine treated patients, those with high levels of uPA:PAI-I complex showed a shorter progression-free survival (PFS) compared to patients with lower uPA:PAI-I complex levels (P=0.035). Furthermore, in the multivariate regression analysis a significant lower rate of response to first-line endocrine therapy was found in patients with high uPA:PAI-I complex levels compared to patients with low uPA:PAI-I complex levels (odds ratio (OR)=0.27, 95% CI, 0.09-0.59, P=0.018), in addition to the predictive impact of the steroid hormone receptor (ER/PgR) status (OR=2.68, 95% CI, 1.08-6.63, P=0.033). Complex levels did not predict efficacy of chemotherapy in patients with advanced breast cancer. The results show that the plasminogen activation system affects the response to endocrine therapy independent of steroid hormone receptor status and may be of help to further refine the indication for this treatment in individual patients. Further studies are warranted to explain this underlying resistance to endocrine therapy when uPA:PAI-I levels are high.

One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system. This system comprises of, among others, the urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-1). In this study we investigated whether the uPA:PAI-1 complex is associated with the responsiveness of patients with primary breast cancer to adjuvant systemic therapy. Quantitative enzyme-linked immunosorbent assays were used to assess the levels of uPA, PAI-1, and uPA:PAI-1 complex in 1119 tumors of patients with primary invasive breast cancer. These patients were followed for a median follow-up time of 59 months (range, 2-267 months) after the primary diagnosis. Correlations with well-known clinicopathological factors, and univariate and multivariate survival analyses were performed. High uPA:PAI-1 complex levels were correlated with an adverse histological grade, and inversely associated with negative estrogen and progesterone receptor status. High tumor levels of uPA:PAI-1 complex predicted an early relapse in the univariate relapse-free survival analysis (P < 0.001). The multivariate analysis showed that high uPA:PAI-1 complex levels were associated with a decreased relapse-free survival time (P = 0.033), independently of age, tumor size, number of lymph nodes affected, progesterone receptor status, uPA, adjuvant endocrine, and chemotherapy. More important, it was demonstrated that there is a larger benefit from adjuvant chemotherapy for patients with higher versus lower tumor levels of uPA:PAI-1 complex. The results of this study imply that the expression of uPA:PAI-1 complex independently predicts the efficacy of adjuvant chemotherapy in patients with primary breast cancer.

Purpose: In recent studies, we showed that TP53 gene mutation or high levels of cytosolic vascular endothelial growth factor (VEGF) in estrogen receptor (ER)-alpha-positive primary breast tumors predict a poor disease outcome for patients treated with first-line tamoxifen for advanced disease. Mutant TP53 may up-regulate VEGF, whereas, on the other hand, wild-type TP53 may decrease VEGF production., Experimental Design: In the present study, we aimed to assess the combined predictive value of TP53 gene mutation and VEGF status of 160 advanced breast cancer patients with ER-positive tumors who were treated with tamoxifen (median follow-up from start of tamoxifen treatment, 64 months). To assess TP53 gene mutation status, the entire open reading frame was sequenced; for VEGF status, an ELISA was used., Results: In univariate analysis, both TP53 gene mutation (28% of the tumors) and a VEGF level above the median value were significantly associated with a short progression-free survival, post-relapse overall survival, and a poor rate of response to tamoxifen. In Cox multivariate regression analysis including the traditional predictive factors, the addition of TP53 gene mutation and VEGF status, alone or in combination, significantly predicted a poor efficacy of tamoxifen treatment. When the two factors were combined, a significantly decreased odds ratio was seen for the rate of response (odds ratio, 0.27). Similarly, an increased hazard ratio (HR) was seen for progression-free survival (HR, 2.32) and post-relapse overall survival (HR, 1.68) in the group with mutant TP53 and high VEGF compared with the group with both risk factors absent., Conclusions: Combined TP53 gene mutation status and high VEGF levels of ER-positive primary breast tumors independently predict a poor course of the disease of patients with advanced breast cancer treated with tamoxifen. These patients, having unfavorable tumor characteristics, might benefit more from other types of (individualized) treatment protocols.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and in several other cancer types. In the present study, we have measured with ELISA the levels of VEGF in cytosolic extracts of 845 primary breast tumors of patients who developed a recurrence during follow-up. All of the patients received tamoxifen (n = 618) or cyclophosphamide, methotrexate, 5-fluorouracil (CMF) or 5-fluorouracil, Adriamycin, cyclophosphamide (FAC) chemotherapy (n = 227) as first-line systemic therapy after diagnosis of advanced disease. VEGF levels were not related to age or menopausal status but were negatively related to the cytosolic levels of estrogen receptor and progesterone receptor (P < 0.0001). In patients who relapsed within 1 year after primary surgery, tumor VEGF levels were higher than in patients who showed a longer disease-free interval (P = 0.0005). In patients with a first relapse in the viscera, VEGF levels were higher compared with those that relapsed to the bone or soft tissue (P = 0.0004). In univariate analysis for response to first-line tamoxifen therapy, patients with high or intermediate levels showed a poor rate of response, compared with patients with low tumor-VEGF levels (P = 0.0001). Similarly, in multivariate analysis for response to tamoxifen treatment, corrected for age, site of relapse, disease-free interval, and estrogen receptor and progesterone receptor status, VEGF status was an independent predictive factor (P = 0.009). In concordance, higher levels of VEGF were associated with a short progression-free survival and postrelapse overall survival (both, P < 0.0001). On first-line chemotherapy, the rate of response decreased with higher tumor levels of VEGF, both in univariate (P = 0.003) and in multivariate analysis (P = 0.004). Furthermore, higher VEGF levels were associated with a short progression-free survival (P = 0.003) and postrelapse overall survival (P = 0.001). In conclusion, the tumor VEGF level is an important independent marker that predicts a poor efficacy of both tamoxifen and chemotherapy in advanced breast cancer. Knowledge of the tumor level of VEGF might be helpful in selecting individual patients who may benefit from treatments with antiangiogenic agents combined with conventionally used drugs.

Using a previously developed enzyme-linked immunosorbent assay (ELISA), the levels of the receptor for urokinase-type plasminogen activator (uPAR) were determined in cytosols and corresponding membrane pellets derived from 878 primary breast tumours. The levels of uPAR in the pellet extracts were more than 3-fold higher than those measured in the cytosols (P< 0.001). Moreover, the uPAR levels in the two types of extracts were weakly, though significantly, correlated with each other (rS= 0.20, P< 0.001). In Cox univariate analysis, high cytosolic levels of uPAR were significantly associated with reduced overall survival (OS) and relapse-free survival (RFS). The levels of uPAR in pellet extracts appeared not to be related with patient survival. In multivariate analysis, elevated levels of uPAR measured in cytosols and pellet extracts were found to be independent predictors of poor OS, not RFS. The prediction of poor prognosis on the basis of high uPAR levels emphasizes its important role in plasmin-mediated degradation of extracellular matrix proteins during cancer invasion and metastasis., (Copyright 2001 Cancer Research Campaign.)