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Objectives: Deviations in haemostasis are found in about 50 % of patients with cancer and up to 90% of those with metastatic disease. Many studies investigate the dynamics of the processes of coagulation and fibrinolysis and their role as a predictor of therapeutic response, early relapse, or metastasis risk., Background: To investigate the serum levels of urokinase plasminogen activator (uPA) in patients with brain metastases treated with robotic stereotactic radiosurgery (SRS) with CyberKnife., Material and Methods: Serum levels of urokinase plasminogen activator (uPA) were measured in 66 patients with solid tumours, divided into two groups, with oligometastatic disease and brain metastases. In this prospective longitudinal study, the serum levels of uPA were measured before starting the therapy and at the first, third, and sixth months after patients were irradiated with the CyberKnife system., Results: Analysis of serum uPA levels in the post-treatment period showed a statistically significant decrease between the baseline and the 6 months post-treatment time point in both patient groups. The baseline value of serum uPA in the group with lung cancer decreased by 62.7 %, and in the group with other types of cancer - by 60 %. Despite the significant reduction of serum uPA levels 6 months after the treatment, the levels remained significantly higher in both groups than in healthy controls., Conclusion: Ongoing research on uPA and cancer will enrich our knowledge and expand the possibilities for clinical utilization of the marker in the oncology setting (Tab. 2, Ref. 18).

Background: Previous studies have showed that the high expression of urokinase plasminogen activator (uPA) in pathology and serology is closely related to the progression of hepatocellular carcinoma (HCC). However, there are no systematic reviews for these evidence, and the association between uPA and HCC is still not completely understood. Therefore, we will undertake a systematic review of the literature to summarize previous evidence regarding this topic, in order to clarify the prognostic significance of uPA in HCC., Methods and Analysis: Studies comparing the HCC patients with high and low expression of uPA on the clinicopathological features and the prognosis are eligible for this review. Outcomes include all endpoints about survival and clinicopathological features. Prospective or retrospective primary studies which published in English will be included. Four databases of Medline, EMBASE, Web of Science, and the Cochrane Library will be systematically searched from their inception to Mar 2021 to retrieve relevant studies. Reference lists of included studies will be manually reviewed and grey literatures will be identified by Google Scholar. Two reviewers will independently screen the records and extract the information and data of the included studies. The Newcastle-Ottawa Scale will be used to assess the quality of included studies. Hazard ratio and 95% confidence interval will be pooled to assess the association between uPA expression and the prognosis. Pooled odds ratio and 95% confidence interval will be used for other outcomes. Heterogeneity will be assessed using the Cochrane Q test and I2 statistic, and a subgroup analysis will be performed if necessary. Grades of Recommendation, Assessment, Development and Evaluation method will be applied to assess the certainty of evidence., Ethics and Dissemination: This protocol required information extracted from previously published articles. So, there is no ethical problem in this study. We plan to publish our findings in peer-reviewed journals and relevant conference proceedings., Systematic Review Registration: This study has been registered with the International Prospective Register of Systematic Reviews database (no.CRD42020150340)., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: Urokinase-plasminogen activator (uPA), its receptor (uPAR), and the plasmin-activator inhibitor type 1 (PAI-1) have been associated with oncologic outcomes in various malignancies and could help identify bladder cancer (BC) patients treated with radical cystectomy (RC) who are likely to benefit from intensification of therapy to prevent disease progression. Our aim was to assess the value of uPA, uPAR, and PAI-1 for prognosticating survival outcomes of patients treated with RC for BC., Materials and Methods: Tumor specimens from 272 consecutive patients treated with RC for advanced BC were assessed with immunohistochemical staining for uPA, uPAR, and PAI-1. Overexpression was assessed by pathological image analysis. Kaplan-Meier estimates and multivariable Cox-regression were used to analyze survival. Harrell's C-index was used to assess for clinical impact of the uPA system., Results: uPA, uPAR, and PAI-1 were overexpressed in 48.2%, 51.1%, and 52.2% of patients, respectively. uPA overexpression was associated with lymphovascular invasion (P = 0.034) and nodal status (P = 0.013); PAI-1 overexpression was associated with primary muscle-invasive BC (P = 0.015) and lymphovascular invasion (P = 0.024). uPA, uPAR, and the number of overexpressed markers were all 3 significantly associated with shorter overall recurrence-free-, distant recurrence-free-, and cancer-specific survival. In multivariable analyses, uPA overexpression remained associated with shorter recurrence-free survival (hazard ratio [HR] = 1.79; P = 0.036) in the entire cohort, in patients without lymph node metastasis (HR = 1.98; P = 0.018) and those with nonorgan-confined disease (HR = 1.98; P = 0.022). uPAR overexpression was associated with shorter recurrence-free survival in patients without lymph node metastasis (HR = 2.01; P = 0.021) and those with organ-confined disease (HR = 4.11; P = 0.037)., Conclusion: Members of the uPA system are associated with features of biologically aggressive BC and oncologic outcomes. However, their value beyond currently available information remains limited., Competing Interests: Disclosure of potential conflicts of interest S.F. Shariat has a consulting or advisory role for the following: Astellas, Astra Zeneca, Bayer, BMS, Cepheid, Ferring, Ipsen, Jansen, Lilly, MSD, Olympus, Pfizer, Pierre Fabre, Richard Wolf, Roche, Sanochemia, Sanofi, Takeda, Urogen., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many studies have shown that urokinase plasminogen activator (uPA) is causally involved in promoting cancer invasion and metastasis. Thus, monitoring uPA levels could be very useful in cancer diagnosis, identification of initial metastasis, and guiding cancer treatment. Here, the authors developed a novel and scalable uPA sensor based on a graphene-gold nanoparticle platform that uses fluorescence of quantum dots to rapidly (<1 h) detect uPA up to 100 pM. Indeed, the authors' sensor is highly selective and showed an ability to sense up to 100 pM uPA even in the presence of complex biological milieu such as the fetal bovine serum.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N -methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

The unraveling of enzymatic reactions, especially identification of enzymatic substrates or products, is important to elucidate biological processes. Here a selenium-isotopic signature for mass spectrometric identification of enzymatic-related species is demonstrated by using selenium-containing peptides (SePeps) as substrates. Thus a strategy is proposed for rapid and precise assay of multiple enzyme activity. These SePeps can be synthesized by introduction of one selenomethionine residue in the sequence and simply identified in the full-scan mode with the feature of distinctive selenium-isotopic distribution without MS/MS verifications, which proposes a novel solution to the specific identification of enzyme-related species, allows to exclude the interferences of species with tiny mass differences in bio-samples, and meanwhile can offer a judgement on data accuracy for the analysis of enzyme activities. As a proof-of-concept, a method for multiple analysis of two representative enzymes in MCF-7 cell lysate has been developed with the isotopic peak areas of either SePep substrates or enzymatic products with the top intensities. These results could be the foundation to extend the method for more complicated enzyme systems. The selenium-isotopic signature provides a powerful protocol for high-throughput assays of peptide-metabolizing enzymes with enhanced confidence and can be extended to screen enzymatic reaction-related substrates., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background uPA and PAI-1 are breast cancer biomarkers that evaluate the benefit of chemotherapy (CT) for HER2-negative, estrogen receptor-positive, low or intermediate grade patients. Our objectives were to observe clinical routine use of uPA/PAI-1 and to build a new therapeutic decision tree integrating uPA/PAI-1. Methods We observed the concordance between CT indications proposed by a canonical decision tree representative of French practices (not including uPA/PAI-1) and actual CT prescriptions decided by a medical board which included uPA/PAI-1. We used a method of machine learning for the analysis of concordant and non-concordant CT prescriptions to generate a novel scheme for CT indications. Results We observed a concordance rate of 71% between indications proposed by the canonical decision tree and actual prescriptions. Discrepancies were due to CT contraindications, high tumor grade and uPA/PAI-1 level. Altogether, uPA/PAI-1 were a decisive factor for the final decision in 17% of cases by avoiding CT prescription in two-thirds of cases and inducing CT in other cases. Remarkably, we noted that in routine practice, elevated uPA/PAI-1 levels seem not to be considered as a sufficient indication for CT for N≤3, Ki 67≤30% tumors, but are considered in association with at least one additional marker such as Ki 67>14%, vascular invasion and ER-H score <150. Conclusions This study highlights that in the routine clinical practice uPA/PAI-1 are never used as the sole indication for CT. Combined with other routinely used biomarkers, uPA/PAI-1 present an added value to orientate the therapeutic choice.

A novel Au-Se nanoprobe with remarkable anti-interference ability for glutathione was developed for real-time in situ monitoring of the upstream and downstream regulatory relationship between uPA and MMP-9 proteins in the pathway.

We retrospectively investigated concordance of EndoPredict (EPclin) with urokinase plasminogen activator and plasminogen activator inhibitor-1 (uPA/PAI-1) in 72 breast cancer patients and compared the results with grading, molecular subtype and chemotherapy recommendation. Compared to uPA/PAI-1, EPclin proved to be more conservative concerning correlation with clinicopathological parameters and was significantly associated with the recommendation of adjuvant chemotherapy., (© 2019 Wiley Periodicals, Inc.)

Herein, we established a novel ultrasensitive photoelectrochemical biosensor for detecting urokinase-type plasminogen activator (u-PA), based on a g-C 3 N 4 /CdS nanocomposite. The prepared nanocomposite was characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, ultraviolet-visible absorption spectroscopy, and Fourier transform infrared spectroscopy, thus indicating that the nanocomposite was prepared successfully. In the typical process, the prepared nanocomposite was deposited on the surface of a bare FTO electrode. After being air-dried, the g-C 3 N 4 /CdS nanocomposite modified electrode was successively incubated with antibody against urokinase-type plasminogen activator and the blocking agent BSA to produce a photoelectrochemical biosensor for u-PA. In the presence of target u-PA antigen, the photocurrent response of the prepared biosensor electrode decreased significantly. The proposed novel photoelectrochemical biosensor exhibited good sensitivity, specificity, and reproducibility for u-PA detection, and a low detection limit of 33 fg mL -1 , ranging from 1 μg mL -1 -0.1 pg mL -1 . The proposed strategy should provide a promising method for detection of other biomarkers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) act in the proteolysis of basement membrane and extracellular matrix structures, facilitating tumor invasion. The purpose of this study was to evaluate the relationship between these proteins and clinicopathological parameters in squamous cell carcinoma of the oral tongue (SCCOT). Sixty cases of SCCOT were submitted to immunohistochemistry and analyzed semiquantitatively at the invasion front and in the tumor core. The results were associated with lymph node metastasis, clinical stage, locoregional recurrence, clinical outcome and histological grade of malignancy. A higher expression of uPA was observed in cases of tumors of high-grade versus low-grade malignancy (p = 0.010). Moreover, the cases with the worst pattern of invasion presented an overexpression of uPA (p = 0.011). The presence of locoregional recurrence was associated with uPAR (p = 0.039), and the expression of both biomarkers was much higher at the invasion front than in the tumor core (p < 0.001). The results suggest uPA and uPAR are involved in the progression and aggressiveness of SCCOT, mainly at the tumor-host interface.

Hard-to-treat cancers are closely relative to uncontrolled cell proliferation, invasion, and metastasis. Assessing proliferation and invasion properties of tumor cells both in vitro and in vivo is especially important for acquiring reliable information for cancer pathogenesis, drug screening, and therapeutic effect evaluation. Herein, we developed a multicolor fluorescent nanoprobe for simultaneously monitoring breast cancer cells' proliferation marker Ki-67 and invasion marker urokinase plasminogen activator (uPA). After treated with the anticancer drugs tamoxifen and curcumin, the changes in cancer cell proliferation and invasion properties were visually detected and therapeutic effects of corresponding drugs were further assessed in vitro and in vivo. The design of the fluorescent nanoprobe opens up an avenue for investigating unscheduled proliferation, invasion, and metastasis in living cells and in vivo and as such will be a promising tool to screen antitumor drugs and evaluate drug efficiency in an extremely efficient manner.

Background: Adjuvant therapy decisions in early breast cancer are based on accurate risk assessment. Urokinase plasminogen activator (uPA) and plaminogen activator inhibitor-1 (PAI-1) have been the first biomarkers in hormone receptor (HR) positive breast cancer to reach highest level of evidence. The EndoPredict test (EPclin) combines gene expression information with nodal status and tumor size. The aim of this prospective study was to compare uPA/PAI-1 and EPclin as prognostic biomarkers with regard to feasibility, risk stratification and impact on adjuvant therapy recommendation., Materials and Method: 395 patients with HR positive, HER2 negative, intermediate risk breast cancer were enrolled. Relations and concordance of histologic grading as well as EPclin and uPA/PAI-1 values were assessed by Spearman's rank correlation coefficient and Cohen's Kappa. To compare decision impact of EPclin and uPA/PAI-1 three independent case discussions were held: One with known uPA/PAI-1 and EPclin results, one blinded to EPclin alone and another one blinded to both EPclin and uPA/PAI-1., Results: EPclin could be determined in all 395 (100%), uPA/PAI-1 in 190 (48%) of the tumor samples. EPclin allocated 250 patients (63%) to the low-risk group and 145 patients (37%) to the high-risk group, whereas uPA/PAI-1 allocated 88 patients (46%) to the low-risk group and 102 patients (54%) to the high-risk group. In 59% of cases, both tests showed concordant results. EPclin resulted more frequently in a change of therapy recommendation than the uPA/PAI-1 test (46% vs 24%). Recommendation of adjuvant chemotherapy (CTX) was abandoned twice as often by EPclin (45%) compared to uPA/PAI-1 (22%)., Conclusion: In this first prospective comparison of EPclin and uPA/PAI-1 we found, that EPclin is superior to uPA/PAI-1 with respect to feasibility and decision impact. This leads to substantial avoidance of adjuvant CTX in endocrine-sensitive, HER2-negative breast cancer. Data collection for patients´ clinical outcome is ongoing.

Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 are key players in cancer invasion and metastasis. Both uPA and PAI-1 have been described as prognostic biomarkers; however, non-invasive methods measuring uPA activity are lacking. We developed an indium-111 ( 111 In)-labelled activity-based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA-conjugated uPA inhibitor was synthesized and radiolabelled with 111 In ([ 111 In]MICA-401), together with its inactive, hydrolysed form ([ 111 In]MICA-402). A biodistribution study was performed in mice (healthy and tumour-bearing), and tumour-targeting properties were evaluated in two different cancer xenografts (MDA-MB-231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [ 111 In]MICA-402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [ 111 In]MICA-401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA-MB-231 and HT-29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA-MB-231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non-invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis., Objectives: We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling., Methods and Results: Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction., Conclusion: Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

Background: This study investigated the cost effectiveness of guideline-recommended (American Society of Clinical Oncology, European Society of Medical Oncology) urokinase plasminogen activator (uPA)/plasminogen activator inhibitor-1 (PAI-1) biomarkers to guide adjuvant chemotherapy decisions for hormone receptor-positive, node-negative early breast cancer patients at intermediate risk of relapse, in France, Germany, and The Netherlands., Methods: uPA/PAI-1 testing was compared to chemotherapy for all patients and to no chemotherapy in two age-related subgroups (35-49 and 50-75 years). A partitioned survival analysis was performed using patient-level data for survival outcomes and secondary sources. Mean quality-adjusted life years (QALYs) and costs were estimated over a lifetime horizon to calculate the incremental net monetary benefit (INMB) at a willingness-to-pay of €50,000/QALY. Uncertainty was explored through bootstrap and probabilistic sensitivity analysis using 5000 replicates., Results: In the 35-49 year age group, INMBs were negative when uPA/PAI-1 testing was compared to chemotherapy for all patients but positive when it was compared to no chemotherapy for the three countries. In the 50-75 year age group, INMBs of uPA/PAI-1 testing compared to both reference strategies were positive in the three countries, with cost-effectiveness probabilities for the uPA/PAI-1 strategy of 65%, 70%, and 59% for France, Germany, and the Netherlands, respectively, compared with chemotherapy for all patients, and 64%, 58%, and 65%, respectively, compared with no chemotherapy., Conclusions: uPA/PAI-1 testing could allow the selection of patients older than 50 years requiring chemotherapy in this population, but the cost effectiveness of this strategy is uncertain. Chemotherapy for all patients is the most cost-effective strategy for patients younger than 50 years., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Rapidly growing cancer cells secrete growth-promoting polypeptides and have increased proteolytic activity, contributing to tumor progression and metastasis. Their presentation in malignant pleural effusion (MPE) and their predictive value for the outcome of pleurodesis and survival were studied., Methods: Between February 2011 and March 2012, MPE samples were prospectively collected from 61 patients. Twenty-five patients with non-malignant pleural effusion in the same period were included as controls. Pleural fluid osteopontin (OPN), vascular endothelial growth factor (VEGF), and urokinase-type plasminogen activator (uPA) concentrations were measured., Results: Patients with MPE had higher pleural fluid OPN, VEGF, and uPA concentrations than those with non-malignant pleural effusion, but only differences in VEGF were statistically significant (p = 0.045). Patients with distant metastases had significantly elevated pleural fluid VEGF concentrations than those without (p = 0.004). Pleural fluid OPN, VEGF, and uPA concentrations were positively correlated in most patients. However, there was no significant difference in pleural fluid OPN, VEGF, and uPA concentrations between patients with successful pleurodesis and those without. There was also no significant difference in cancer-specific survival between sub-groups with higher and lower pleural fluid OPN, VEGF, or uPA concentrations. Patients with successful pleurodesis had significantly longer cancer-specific survival than those without (p = 0.015)., Conclusions: Pleural fluid OPN, VEGF, and uPA concentrations are elevated in MPE but are not satisfactory predictors of pleurodesis outcome or survival. Patients with higher pleural fluid VEGF concentration have higher risk of distant metastasis. Evaluating the benefits of therapy targeting the VEGF pathway in these patients warrants further studies.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and its prognosis remains poor due to the high risk of tumor recurrence and metastasis. Berberine (BBR) is a natural compound derived from some medicinal plants, and accumulating evidence has shown its potent anti-tumor activity with diverse action on tumor cells, including inducing cancer cell death and blocking cell cycle and migration. Molecular targets of berberine involved in its inhibitory effect on the invasiveness remains not yet clear. In this study, we identified that berberine exhibits a potent inhibition on the invasion and migration of HCC cells. This was accompanied by a dose-dependent down-regulation of expression of Cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 in berberine-treated HCC cells. Furthermore, berberine inactivated p38 and Erk1/2 signaling pathway in HCC cells. Primarily, this may be attributed to the up-regulation of plasminogen activator inhibitor-1 (PAI-1), a tumor suppressor that can antagonize uPA receptor and down-regulation of uPA. Blockade of uPA receptor-associated pathways leads to reduced invasiveness and motility of berberine-treated HCC cells. In conclusion, our findings identified for the first time that inactivation of uPA receptor by up-regulation of PAI-1 and down-regulation of uPA is involved in the inhibitory effect of berberine on HCC cell invasion and migration.

Purpose: To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer., Methods: A literature search and prospectively defined study selection sought systematic reviews, meta-analyses, randomized controlled trials, prospective-retrospective studies, and prospective comparative observational studies published from 2006 through 2014. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert panel members used informal consensus to develop evidence-based guideline recommendations., Results: The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that met guideline criteria for clinical utility were found to guide choice of specific treatments or regimens., Recommendations: In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2, the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX, EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens. Treatment decisions should also consider disease stage, comorbidities, and patient preferences., (© 2016 by American Society of Clinical Oncology.)

To investigate the role of suPAR in patients with sepsis admitted to the Emergency Department (ED). We performed multicentre prospective trial including patients admitted to the ED of three different Italian hospitals. Patients were studied upon admission on day 1, 2, 4 and 7. They were subdivided into two groups: sepsis (group 1) and severe sepsis or septic shock (group 2). The two groups were comparable for age, gender and CRP level on day 1. Patients with severe sepsis or septic shock displayed significantly higher baseline levels of suPAR, PCT and lactate. In both groups, suPAR decreased across the time (p < 0.0005). Group 1 was not different from group 2 (p = 0.545) in mortality at 7 days, while group 2 had higher mortality at 30 days than group 1 (p = 0.022). At the multivariate analysis, lactate1 (p = 0.012) and age (p = 0.019) were independent predictors of mortality at 7 days, whereas suPAR1 (p = 0.023) and age (p = 0.032) were independent predictors of mortality at 30 days. Lactate and suPAR resulted the most predictive biomarkers in the risk stratification of patients with suspected infection initially admitted to the ED, according to their role in predicting 7- and 30-day mortality, respectively.

Background: Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) have been shown to be of merit as biomarkers for a variety of cancers. Prostate tissue resections from patients with prostate cancer (PCa) and benign prostatic hyperplasia were analysed to determine the influence of freeze-drying on the recovery of uPA and PAI-1 and their predictive performance., Methods: Prostate tissue was frozen in liquid nitrogen and homogenised into a fine powder in a precooled stainless steel punch homogeniser. One aliquot of the powder was extracted directly, and a second aliquot was freeze-dried overnight and then extracted. The extracts were analysed by FEMTELLE assay to determine the concentrations of uPA and PAI-1. uPA/PAI-1 ratios were calculated for each sample, and the mean ratios for the frozen and the lyophilised tissue were compared., Results: The concentrations of uPA measured for the frozen and lyophilised samples are strongly correlated (R = 0.90 ± 0.05). The same applies to the PAI-1 measured (R = 0.89 ± 0.03). The uPA/PAI-1 ratios for the lyophilised and frozen samples were strongly correlated. The uPA/PAI-1 ratios for frozen and lyophilised samples were found to be essentially the same with values of 0.0344 ± 0.0066 and 0.0340 ± 0.0068, respectively (P = 0.9633)., Conclusion: The recovery of uPA and PAI-1 from a deep frozen prostate tissue homogenate followed by freeze-drying proceeds with a loss of 10 and 11%, respectively, with no influence on the uPA/PAI-1 ratio. Lyophilisation is a safe procedure for the preservation of frozen prostate tissue samples as it permits recovery of the markers without compromising their use for diagnostic purposes., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Urokinase (uPA, urinary plasminogen activator) is a serine protease belonging to the peptidase S1 family. Specifically, uPA cleaves the zymogen plasminogen into the active form (plasmin), which then degrades the fibrin clots. It is widely used as a fibrinolytic agent in thrombolytic therapy and it is also used clinically as a thrombolytic agent. It can be administered to improve the drainage of complicated pleural effusions and empyemas and it is the most effective drug in myocardial infarction. The enzyme was originally identified in human urine for its ability to catalyze the transformation of plasminogen into its active form (plasmin), which degrades fibrin and extracellular matrix components. The present report deals with the analysis and characterization of this preparation.

Biomarkers play an important role in the detection and management of patients with breast cancer. Thus, BRCA1/2 mutation testing is used for risk assessment in families with a high prevalence of breast and ovarian cancer. Following a diagnosis of breast cancer, measurement of multi-analyte profiles such as uPA/PAI-1 or Oncotype DX may be used for determining prognosis and identifying lymph node-negative patients who may be spared from having to receive adjuvant chemotherapy. Other -gene tests such as the PAM50 ROR, Breast Cancer Index, and EndoPredict have been reported to predict the development of late recurrences and thus may be of value in selecting patients for extended hormone therapy. Mandatory assays include estrogen receptors for identification of endocrine-sensitive cancers and HER2 in selecting patients for treatment with anti-HER2 therapy (e.g., trastuzumab, lapatinib, pertuzumab, and ado-trastuzumab emtansine). Finally, serum biomarkers such as CA 15-3 or CEA may be used in monitoring therapy in patients with advanced disease receiving systemic therapy. Promising new biomarkers undergoing evaluation include circulating tumor cells and circulating tumor-derived DNA., (© 2015 Elsevier Inc. All rights reserved.)

Members of the plasminogen-plasmin (PP) system participate in many physiologic functions. In particular, uPA, its receptor (uPAR) and its inhibitor PAI-1 play an important role in cell migration, cell proliferation and tissue remodeling. Through a number of interactions, these components of the PP system are also involved in the pathogenesis of many diseases. In cancer, they modulate the essential processes of tumor development, growth, invasion and metastasis as well as angiogenesis and fibrosis. Thus, quantification of uPA, uPAR and PAI-1 in tumors and, in some cases in the circulating blood, became of potential value in the prognostication of many types of cancer. These include cancer of the breast, stomach, colon and rectum, esophagus, pancreas, glioma, lung, kidney, prostate, uterine cervix, ovary, liver and bone. Published data are reviewed in this chapter. Clinical validation of the prognostic value has also been made, particularly in cancer of the breast. Inclusion of these biomarkers in the risk assessment of cancer patients is now considered in the risk-adapted management in carcinoma of the breast. Factors limiting its broader use are discussed with suggestions how these can be overcome. Hopefully the use of these biomarkers will be applied to other types of cancer in the near future.

Objective: Most endometrial cancers are detected early and have a good prognosis, while some endometrial cancers are highly invasive, metastasize early, and respond suboptimally to therapy. Currently, appropriate model systems to study the aggressive nature of these tumors are lacking. The objective of this study was to establish a mouse xenograft model of endometrial tumors derived from patients in order to study the biological aggressive characteristics that underlie invasion and metastasis., Methods: Endometrial tumor tissue fragments (1.5 mm × 1.5 mm) from patients undergoing surgery, were transplanted under the renal capsule of NOD scid gamma mice. After 6-8 weeks, tumors were excised and serially transplanted into additional mice for propagation. Immunohistochemical analysis of the tumors was done for various tumor markers., Results: Four cases of different subtypes of endometrial cancer were grown and propagated in mice. Three of the four tumor cases invaded into the kidneys and to adjacent organs. While all tumors exhibited minimal to no staining for estrogen receptor α, progesterone receptor staining was observed for tumor grafts. In addition, levels and localization of E-cadherin, cytokeratin and vimentin varied depending on subtype. Finally, all tumor xenografts stained positively for urokinase plasminogen activator while 3 tumor xenografts, which showed invasive characteristics, stained positively for urokinase plasminogen activator receptor., Conclusion: Endometrial tumors transplanted under the renal capsule exhibit growth, invasion and local spread. These tumors can be propagated and used to study aggressive endometrial cancer.

Background: Members of the urokinase-type plasminogen activator (uPA) system including uPA, its receptor uPAR and the plasminogen activator inhibitor 1 (PAI-1) play an important role in tumour invasion and progression in a variety of tumour types. Since the majority of clear cell renal cell carcinoma (ccRCC) shows distant metastasis at time of diagnosis or later, the interplay of uPA, uPAR and PAI-1 might be of importance in this process determining the patients' outcome., Methods: Corresponding pairs of malignant and non-malignant renal tissue specimens were obtained from 112 ccRCC patients without distant metastasis who underwent tumour nephrectomy. Tissue extracts prepared from fresh-frozen tissue samples by detergent extraction were used for the determination of antigen levels of uPA, uPAR and PAI-1 by ELISA. Antigen levels were normalised to protein concentrations and expressed as ng per mg of total protein., Results: Antigen levels of uPA, uPAR, and PAI-1 correlated with each other in the malignant tissue specimens (rs=0.51-0.65; all P<0.001). Antigen levels of uPA system components were significantly higher in tissue extracts of non-organ confined tumours (pT3+4) compared to organ-confined tumours (pT1+2; all P<0.05). Significantly elevated levels of uPAR and PAI-1 were also observed in high grade ccRCC. When using median antigen levels as cut-off points, all three uPA system factors were significant predictors for disease-specific survival (DSS) in univariate Cox's regression analyses. High levels of uPA and uPAR remained independent predictors for DSS with HR=2.86 (95% CI 1.07-7.67, P=0.037) and HR=4.70 (95% CI 1.51-14.6, P=0.008), respectively, in multivariate Cox's regression analyses. A combination of high antigen levels of uPA and/or uPAR further improved the prediction of DSS in multivariate analysis (HR=14.5, 95% CI 1.88-111.1, P=0.010). Moreover, high uPA and/or uPAR levels defined a patient subgroup of high risk for tumour-related death in ccRCC patients with organ-confined disease (pT1+2) (HR=9.83, 95% CI 1.21-79.6, P=0.032)., Conclusions: High levels of uPA and uPAR in tumour tissue extracts are associated with a significantly shorter DSS of ccRCC patients without distant metastases.

Context and Aims: Breast cancer prognosis and predictive biomarkers development would allow sparing some patients from chemotherapy or identifying patients for whom chemotherapy would be indicated. In this context, in 2009, the French National Cancer Institute, a National Health and Science Agency dedicated to cancer, in collaboration with the French society of senology and breast pathology (SFSPM) published a report on the assessment of the prognostic and the predictive clinical validity of tissular biomarkers, uPA/PAI-1, Oncotype DX™ and MammaPrint(®), in breast cancer management. They concluded that only the uPA/PAI-1 prognosis value reached the highest level of evidence (LOE I according to Hayes 1998 classification). In 2012, it was decided to update this report since new data have emerged and because information disparities among clinicians have been identified. This article aims to present the main conclusions together with the levels of evidence associated with those conclusions., Methods: The updating process was based on literature published since 2009 appraisal and on multidisciplinary and independent experts' opinion. The levels of evidence (LOE) used are those of the classification defined by Simon in 2009 (updated Hayes 1998 classification): LOE IA and LOE IB: high level of evidence; LOE IIB and LOE IIC: intermediate level of evidence; LOE IIIC and LOE IV-VD: low level of evidence., Conclusions: Among patients without lymph-node involvement, uPA/PAI-1, invasion process biomarkers, reach the highest level of evidence for 10 years recurrence free survival prognosis (LOE IA according to Simon). The predictive value to anthracyclins chemotherapy remains to be confirmed. Oncotype DX™ and MammaPrint(®) prognosis and predictive value do not reach the LOE I level. This updating' process confirms the 2009 levels of evidence for all the three biomarkers prognosis value. Besides, concerning Oncotype DX™ and MammaPrint(®), new data do not allow to conclude neither to their complementary clinical information to other clinicopathological existing biomarkers nor to a favorable cost-efficiency ratio in therapeutic decision making and this because of the methodological weakness and uncertainty that are identified in the selected studies. Practically, beyond the prognosis and predictive biomarkers validity, the clinical utility of a new biomarker for chemotherapy indication depends on its clinical added information with regard to validated biomarkers (HR, HER2 and Ki67) and to clinicopathological parameters. Since they are the sole validated biomarkers of the invasion process, uPA/PAI-1 could complete clinical information of other clinicopathological factors and consequently could confer an added clinical value. However, data concerning the impact of this information on chemotherapy clinical indication are lacking., (Copyright © 2014. Published by Elsevier Masson SAS.)

Unlabelled: Urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) are key factors for tumor invasion and development of metastases in breast cancer. Prospective studies confirmed the prognostic significance of these factors for development of distant metastases. The predictive impact of uPA and PAI-1 for metastatic sentinel lymph node involvement is unclear., Patients and Methods: Between 2006 and 2008 uPA and PAI-1 were measured in 184 out of 1,035 patients for primary breast cancer. uPA and PAI-1 were analyzed with an ELISA assay. Measured concentrations were considered as negative for uPA <3 ng/ml and for PAI-1 <14 ng/ml., Results: In a retrospective analysis, 173/184 women had a negative sentinel lymph node and 11/184 women had a metastatic sentinel lymph node. From the 11 women with a positive sentinel lymph node 7 had elevated values for uPA and 4 had elevated values for PAI-1. Four and 7 women were uPA- and PAI-1-negative, respectively. Sensitivity, specificity, positive and negative predictive values for uPA were 63.3%, 50.9%, 7.6%, 95.6% and for PAI-1 36%, 52.6%, 4.7%, 92.9%. Even the combination of both uPA and PAI-1 values did not detect 3/11 women with metastatic lymph node involvement., Conclusion: uPA and PAI-1 alone or in combination did not identify all patients with metastatic lymph node involvement. Thus, uPA and PAI-1 cannot be considered as predictive selection parameters to avoid sentinel lymph node biopsy in case of negative values for uPA or PAI-1., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aims: The purpose of this study was to investigate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor (PAI)-1 on podocytes in immunoglobulin A (IgA) glomerulonephritis (GN)., Methods: Renal biopsy specimens from 52 IgA GN patients were deparaffinized and subjected to immunohistochemical staining for uPA, PAI-1, and uPAR. The biopsies were classified into three groups according to the expression of uPA and uPAR on podocytes: uPA, uPAR, and a negative group. The prevalences of the variables of the Oxford classification for IgA GN were compared among the groups., Results: On podocytes, uPA was positive in 11 cases and uPAR was positive in 38 cases; by contrast, PAI-1 was negative in all cases. Expression of both uPA and uPAR on podocytes was less frequently accompanied by tubulointerstitial fibrosis., Conclusions: Our results suggest a possible protective effect of podocyte uPA/uPAR expression against interstitial fibrosis.

Urokinase-type plasminogen activator receptor (uPAR) regulates pericellular proteolysis by binding the serine proteinase urokinase-type plasminogen activator (uPA) that promotes cell surface activating of plasminogen to plasmin. In addition, uPAR as a glycosylphosphatidylinositol (GPI)-anchored signaling receptor affects cell migration, differentiation, and proliferation. The aim of the present study was to monitor the occurrence and distribution pattern of uPAR in cells of the rat molar tooth germ. By means of immunocytochemistry moderate, uPAR immunoreactivity was detected in epithelial cells of the enamel organ and in ameloblasts and odontoblasts. RT-PCR and Western blotting experiments demonstrated the expression of uPAR in phorbol 12-myristate 13-acetate (PMA)-stimulated dental epithelial cells (HAT-7 cells). A substantial part of uPAR was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. However, co-immunoprecipitation experiments showed that uPAR and caveolin-1 do not associate with each other directly. Cell stimulation experiments with PMA indicated that protein kinase C (PKC)-mediated signaling pathways contribute to the expression of uPAR in cells of the enamel organ. The localization of uPAR in membrane rafts provides a basis for further investigations on the role of uPAR-mediated signaling cascades in ameloblasts.

The determination of the invasion markers urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) has further improved the possibilities for individualized therapy of breast cancer. To date, quantitative measurement by ELISA, that needs large amounts of fresh, frozen material, is the only standardized procedure for diagnostic purposes. Therefore, the aim of this study was the establishment of a reliable alternative method based on immunohistochemistry (IHC) and image analysis requiring only small amounts of fixed tumor tissue. Protein expression of uPA and PAI-1 was analyzed in HOPE-fixed tumor samples using tissue microarrays (TMAs) and semiquantitative image analysis. The results of both methods were significantly correlated and risk assessment showed an overall concordance of 78% (83/107; high- and low-risk) and of 94% (74/79) regarding only high-risk patients. The data demonstrate that optimized IHC in combination with image analysis can provide adequate clinical significance compared to ELISA-derived determination of uPA and PAI-1., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

Background: Vessels heal after injury and G protein-coupled receptors are involved in the vascular smooth muscle cell proliferation required to form intimal hyperplasia. We have previously identified the role of Gαq in vascular smooth muscle cell proliferation in vitro. This study now examines the role of Gαq in the developing intimal hyperplasia in a murine model and the impact of disruption of Gαq signaling on intimal hyperplasia development., Methods: We employed a murine femoral wire injury model in which a micro-wire is passed through a branch of the femoral artery and used to denude the common femoral artery. We perfusion-fixed specimens and stained sections with hematoxylin-eosin and Movat's stains such that morphometric analysis could be performed using an Image-Pro system. We also harvested additional specimens of femoral artery and snap-froze them for Western blotting or zymography, to allow for the study of G protein expression and both protease expression and activity. We used contralateral vessels as controls. We immersed additional vessels in pluronic gel containing the chemical Gαq G protein inhibitors GP-2A, siRNA to Gαq or adenovirus containing mutant inactive Gαq., Results: Gαq expression increased in a time-dependent manner after femoral artery injury. Sham-operated vessels did not produce such a response. Inhibition of Gαq reduced cell proliferation without affecting cell migration. Interruption of Gαq signaling also inhibited the development of intimal hyperplasia. Inhibition of Gαq did not alter peak urinary-type plasminogen activator activity and expression, but did increase early plasminogen activator inhibitor-1 activity and expression. Inhibition of Gαq reduced peak metalloproteinase (MMP)-9 activity at Day 3 but did not influence peak MMP-2 activity at Day 7. Protein expression for MMP-9 was also decreased, but that of MMP-2 was not affected. There were no changes in the expression or the activity of the respective inhibitors for MMP-9 and MMP-2, and tissue inhibitor of metalloproteinases-1 and -2., Conclusions: These data demonstrate that femoral wire injury in the mouse is associated with a time-dependent increase in Gαq expression. Inhibition of Gαq alters cell proliferation and is associated with decreased MMP-9 expression and activity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Fibrinolysis (plasmin-mediated cleavage of fibrin structures) is a process in which fibrin clots can be removed from blood vessels, allowing the return of normal vascular function. Although several methods have been developed to measure plasmin activity and plasminogen (the plasmin precursor) concentrations, they are only moderately sensitive and quantitative and require large amounts of reagents, limiting their applicability. We developed two simple, label-free homogeneous assays using gold nanoparticles (Au NPs) for detection of fibrinolysis-related proteins and their activator (urokinase that converts plasminogen to plasmin) and inhibitor (α2-plasmin inhibitor that inhibits plasmin and plasminogen bound to fibrin). We used a fibrinolysis-based sensor, based on plasmin-mediated cleavage of fibrinogen-modified Au NPs (Fib-Au NPs) leading to aggregation of Au NPs, to determine plasmin activity in a biological medium mimic solution. A combination of thrombin (Thr) and Fib-Au NPs allowed us to analyze plasmin activity and plasminogen concentrations in serum through Thr-induced agglutination of Fib-Au NPs. The limit of detection (LOD; S/N=3) of this sensor for plasmin in serum was 0.4 nM (ca. 1.7×10(-4) unit mL(-1)). These label-free assays offer several advantages over conventional assays, including allowing rapid and simple readings with the naked eye or measurement by UV-vis absorption spectroscopy., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Invasion factors uPA/PAI-1 are guideline-recommended (ASCO, AGO) biomarkers for decision support regarding adjuvant chemotherapy (CTX) in women with primary breast cancer. They define a high-risk group with strong benefit from adjuvant CTX and a low-risk group with uncertain benefit and excellent survival without CTX. In a target population (age > 35/N0/G2/HR+/HER2-), administration of adjuvant CTX is not mandatory in Germany and other countries. Based on existing data, this economic model was developed to determine for the first time health economic impact of uPA/PAI-1 testing. Incremental cost-effectiveness ratio (ICER) resulting from uPA/PAI-1 testing was estimated for the target population by Markov modeling and sensitivity analysis. Survival data, CTX-uPA/PAI-1 interactions, and uPA/PAI-1 hazard ratios were derived from the Chemo N0 trial and other evidence. Incremental costs were computed from a payer's perspective appropriate to the German setting. Incremental effectiveness in life years (ly) was estimated taking into account age-adjusted life expectancy, disease-free survival (with/without CTX), and 2 years post-relapse survival. Sensitivity analysis was performed by varying residual adjuvant CTX benefit in the low-risk group, denoted HR&#95;CTX(LR), in range 0.8-0.99. All patients receive adjuvant endocrine therapy. Test is restricted to patients willing to forgo CTX if both markers are below specific cut-off values and to undergo CTX otherwise. For a typical 55-year-old patient, comparing to an "all-CTX" strategy without the test, ICER (all-CTX vs. test) > 50,000 if HR&#95;CTX(LR) > 0.85, with savings of 18,500 per low-risk patient attributable to the test. The cost-effectiveness of forgoing CTX is very high as HR&#95;CTX(LR) approaches one. Conversely, comparing to a "no-CTX" strategy (e.g., patients who initially refuse CTX) without the test, the test is very cost-effective at all ages in the target group if high-risk patients are willing to undergo CTX: ICER (test vs. no-CTX) < 6,000 at age 55 and even better at younger ages, remaining < 25,000 up to age 75. The main determinants of cost utility are age and residual CTX benefit in low-uPA/PAI-1 patients. The uPA/PAI-1 test is cost-effective in the target group compared to either an "all-CTX" or a "no-CTX" scenario. This model thus lends health economic support to current guideline recommendations that uPA/PAI-1 testing is beneficial for BC patients with no lymph node involvement.

Study Design: The immunohistochemical profile of nuclear factor-κ B (NF-κB)/p50, NF-κB/p65, matrix metalloproteinase (MMP)-9, MMP-2, and urokinase-type plasminogen activator (u-PA) proteins was examined in spinal cord tissues coming from rabbits, which underwent chronic cervical spinal cord compression., Objective: To study the potential role of NF-κB and extracellular matrix proteins under the chronic mechanical compression of the cervical spinal cord., Summary of Background Data: Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord dysfunction among adults older than 55 years. Neuronal loss, myelin destruction, axonal degeneration, and glial scar formation are the principal neuropathological features of CSM. However, the biologic pathways that lead to these features remain unclear., Methods: In this study, we used a new animal experimental model of CSM developed in our laboratory. Briefly, after posterior cervical laminectomy, gradual and progressive compression (during 20 weeks) was achieved by introducing a piece of aromatic polyether (0.07 mm thick) under the C6 lamina in 15 New Zealand rabbits. In control animals (n = 15), the aromatic polyether was implanted and then removed after 60 seconds (sham operation). The immunoreactivity of p50 and p65 subunits of NF-kB, as well as that of MMP-2, MMP-9, and u-PA, was evaluated in paraffin-embedded spinal cord sections coming from both groups. The evaluation was performed using immunohistochemistry technique and the results were analyzed using SPSS for Windows, release 12.0 (SPSS Inc., Chicago, IL)., Results: Increased immunoreactivity of both NF-κB subunits, p50 and p65, as well as MMP-2, MMP-9, and u-PA was demonstrated in animals with CSM in comparison with controls. Statistical analysis of the results revealed strong positive correlation between NF-κB subunits immunoreactivity and that of MMP-9, MMP-2, and u-PA., Conclusion: There is a strong correlation between the immunoexpression of NF-κB/p50, NF-κB/p65, MMP-2, MMP-9, u-PA, and CSM.

The level of active urokinase (uPA) is decreased in lung fluids of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) whereas α(2)-macroglobulin (α(2)-M), a plasma proteinase inhibitor, is a major component of these fluids. Since there have been reports describing the ability of α(2)-M to form complexes with uPA in vitro, we hypothesized that α(2)-M may interact with uPA in the lung to modulate its biological activity. Pulmonary edema fluids and lung tissues from patients with ALI/ARDS were evaluated for the presence of uPA associated with α(2)-M. Complexes between α(2)-M and uPA were detected in alveolar edema fluids as well as in lungs of patients with ALI/ARDS where they were located mainly in close proximity to epithelial cells. While uPA bound to α(2)-M retains its amidolytic activity towards low-molecular-weight substrates, it is not inhibited by its main physiological inhibitor, plasminogen activator inhibitor 1. We also investigated the functional consequences of formation of complexes between uPA and α(2)-M in vitro. We found that when α(2)-M:uPA complexes were added to cultures of human bronchial epithelial cells (BEAS-2B), activation of nuclear factor-κB as well as production of interleukin-6 and -8 was substantially suppressed compared with the addition of uPA alone. Our findings indicate for the first time that the function of uPA in patients with ALI/ARDS may be modulated by α(2)-M and that the effects may include the regulation of the fibrinolytic and signaling activities of uPA.

We examined whether the expression and activation of pro-matrix metalloproteinase (MMP)-1 varies from that of pro-MMP-13 in the joint fluid of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. To do this, joint fluid was collected from 34 RA and 34 OA patients. The collagenase (pro-MMP-1 and MMP-13, total MMP-1, and MMP-13), gelatinase (total MMP-2 and MMP-9), stromelysin (total MMP-3), matrilysin (total MMP-7), uPA, and tissue inhibitor of MMP (TIMP) levels were measured by ELISA. The level of total MMP-1 in RA joint fluids was similar to that of the OA joint fluid. In contrast, the level of total MMP-13 in the RA group was significantly higher than that of the OA group. Among various MMPs (MMP-2, MMP-3, MMP-7, and MMP-9), only MMP-9 was strongly associated with total MMP-13 in both RA and OA. The level of uPA was also strongly associated with MMP-13 in RA but not OA, while the level of TIMP-1 and TIMP-2 was not significantly different between RA and OA. In conclusion, MMP-9 and uPA might be involved in the activation of pro-MMP-13 through unknown mechanisms in arthritic diseases.

Background: Herein we present the results of two related investigations. The first study determined if concentrations in breast nipple discharge (ND) of two proteins (urinary plasminogen activator, uPA and its inhibitor, PAI-1) predicted the presence of breast atypia and cancer in pre- and/or postmenopausal women requiring surgery because of a suspicious breast lesion. The second study assessed if these proteins increased the predictive ability of a carbohydrate (Thomsen Friedenreich, TF) which we previously demonstrated predicted the presence of disease in postmenopausal women requiring surgery., Methods: In the first study we prospectively enrolled 79 participants from whom we collected ND, measured uPA and PAI-1 and correlated expression with pathologic findings. In the second study we analyzed 35 (uPA and PAI-1 in 24, uPA in an additional 11) ND samples collected from different participants requiring breast surgery, all of whom also had TF results., Results: uPA expression was higher in pre- and PAI-1 in postmenopausal women with 1) cancer (DCIS or invasive) vs. either no cancer (atypia or benign pathology, p = .018 and .025, respectively), or benign pathology (p = .017 and .033, respectively); and 2) abnormal (atypia or cancer) versus benign pathology (p = .018 and .052, respectively). High uPA and PAI-1 concentrations and age were independent predictors of disease in premenopausal women, with an area under the curve (AUC) of 83-87% when comparing diseased vs. benign pathology. uPA, TF, and age correctly classified 35 pre- and postmenopausal women as having disease or not 84-91% of the time, whereas combining uPA+PAI-1+TF correctly classified 24 women 97-100% of the time., Conclusions: uPA and PAI-1 concentrations in ND were higher in women with atypia and cancer compared to women with benign disease. Combining uPA, PAI-1 and TF in the assessment of women requiring diagnostic breast surgery maximized disease prediction. The assessment of these markers may prove useful in early breast cancer detection.

Background: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin., Methods: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry., Results: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions., Conclusions: The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases., (© 2011 John Wiley & Sons A/S.)

Background/aims: Urokinase (uPA) is a serine protease, which together with uPAR, tPA, PAI 1 and PAI 2 forms the plasminogen activator system, a component of metastatic cascade contributing to the invasive growth and angiogenesis of malignant tumours., Methodology: Both preceding therapy and after 6-8 weeks of the treatment, plasma PAI 1 levels (photometric microplate method on the ELISA) and uPA, uPAR, PAI 1 and PAI 2 tissue expression (immunohistochemical reaction) were analysed from 80 colorectal carcinoma patients., Results: Analysis showed higher pre-treatment plasma levels of PAI 1 in patients with advanced tumours, which decreased after surgery or the start of therapy (p=0.004); Patients with higher plasma level PAI 1 before (0.013) and after therapy (0.004) had significantly shorter survival. There was a higher expression of uPA (p<0.001), uPAR (p<0.001), PAI 1 (p=0.042) and PAI 2 (p<0.001) in advanced colorectal carcinoma. A relationship between PAI 2 (p=0.010) and uPAR (p=0.019) expression and survival was demonstrated. There is a correlation between pre-treatment plasma PAI 1 levels and PAI 2 (p=0.028) and uPAR (p=0.043) expression., Conclusions: Immunohistochemical analysis of PAS in tumour tissue and plasma PAI 1 levels was found to be a useful prognostic factor in colorectal carcinoma patients. Plasma PAI 1 could be advantageous in evaluating the effectiveness of a mode of treatment.

Purpose: The intercellular adhesion molecule (ICAM)-1 is expressed on many cell types including endothelial cells and different cancer cell entities. Experimental data strongly indicate that ICAM-1 can activate intracellular signalling pathways in cancer cells leading to enhanced cell motility, invasion and metastasis. Yet, little is known about the role of ICAM-1 expression during malignant progression in breast cancer patients., Methods: We investigated ICAM-1 protein and mRNA expression in two partly overlapping cohorts of breast cancer patients. ICAM-1 protein was detected by Western blot analysis in 104 cases and verified by immunohistochemistry. Additionally, ICAM-1 mRNA microarray data from 169 tumours were analysed., Results: With both methods, high ICAM-1 expression was significantly associated with a poorly differentiated phenotype, a negative estrogen receptor (ER) status and positive lymph node involvement. In addition, there was a significant prognostic impact of ICAM-1 protein overexpression on recurrence-free survival (HR = 2.82, P = 0.023), which was most pronounced in ER-negative tumours. ICAM-1 mRNA overexpression was associated with high urokinase plasminogen activator (uPA) and uPA-inhibitor protein 1 (PAI 1) protein and mRNA levels as well as high Ki67 protein and vascular endothelial growth factor (VEGF) mRNA expression., Conclusions: In our group of patients, ICAM-1 expression was associated with a more aggressive tumour phenotype. Because of its association with malignant progression, ICAM-1 might represent a new target in the treatment of breast cancer patients.

Urokinase plasminogen activator (uPA) has been associated with aggressive behaviour and poor prognosis in human breast cancer, but there is no information on its expression in canine mammary tumours (CMT). uPA immunohistochemical expression was studied in 119 CMT (24 benign, 95 malignant) to investigate its relationship with clinical and histopathological parameters. Dogs with malignant mammary tumours (MMT) underwent a 2-year follow-up evaluation. MMT expressed significantly more uPA than benign tumours. In MMT, high uPA stromal expression was significantly associated with larger tumour size, high Ki-67 expression, invasive growth, high histological grade, regional lymph node metastases, development of distant metastases, and lower overall survival (OS) and disease-free survival (DFS). On the basis of these results, uPA could be considered a useful prognostic factor in dogs with MMT., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aims and Background: Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) are key molecules in pericellular proteolysis, a process that plays an important role in tumor invasion and metastasis. In the current study we investigated the prognostic significance of uPA and PAI-1 in primary invasive breast cancer., Methods and Study Design: uPA and PAI-1 antigen levels were determined by enzyme-linked immunosorbent assay in cytosols of 177 invasive ductal carcinoma specimens. The prognostic significance of uPA and PAI-1 was assessed for overall survival. The median follow-up time was 90 months., Results: In univariate analysis, both uPA (third versus first tertile range of values; P = 0.02; HR = 2.08) and PAI-1 (third versus first tertile; P = 0.0007; HR = 3.1) were significant prognostic markers for overall survival. In multivariate analysis only nodal status (N2 vs N0; P = 0.0001; HR = 3.94) and PAI-1 (third versus first tertile; P = 0.004; HR = 3.05) remained significant independent prognostic factors. Both uPA and PAI-1 were correlated with established prognostic markers including histological grade, tumor size and Nottingham index., Conclusion: Our study with a 7.5-year follow-up confirmed the relation between elevated uPA and PAI-1 values and an aggressive course of invasive breast cancer. The prognostic significance of PAI-1 as an independent marker was proved for the overall group of breast cancer patients and the subgroup of node-positive patients.

Background: Tumor-related proteases such as urokinase-type plasminogen (uPA), plasminogen activator inhibitor (PAI-1), and cathepsin D (cath-D) are involved in the prognosis of breast cancer (BC) and promote the metastatic process. We investigated the relationship between these proteases and their prognostic significance according to the ER status., Methods: uPA, PAI-1, and cath-D levels were prospectively measured in tumor from 316 patients with primary BC. The distribution and relationship between these proteases and ER subsets were analyzed as well as the prognostic significance., Results: Quantitative levels of uPA, and PAI-1 were higher in ER-patients (p< 0.001) whereas no difference was observed for cath-D levels in ER subsets (p = 0.96). In patients with a positive and highly positive ER status, a high cath-D level was associated with a poorer prognosis. Patients in the highly positive ER group experienced poorer survival in the group with a high PAI-1 level compared to the group with a low PAI-1 level. In ER+ patients, cath-D (p= 0.02) and the tumor size (p= 0.03) were independent factors for OS., Conclusion: The prognostic significance of proteases is modulated by the ER status. Cath-D and PAI-1 could identify a high-risk group with an adverse outcome in ER+ patients.

Albumin dialysis using the molecular adsorbent recirculating system (MARS) is a new therapeutic approach for liver diseases. To gain insight into the mechanisms involved in albumin dialysis, we analyzed the peptides and proteins absorbed into the MARS strong anion exchange (SAX) cartridges as a result of the treatment of patients with cholestasis and resistant pruritus. Proteins extracted from the SAX MARS cartridges after patient treatment were digested with two enzymes. The resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry. We identified over 1,500 peptide sequences corresponding to 144 proteins. In addition to the proteins that are present in control albumin-derived samples, this collection includes 60 proteins that were specific to samples obtained after patient treatment. Five of these proteins (neutrophil defensin 1 [HNP-1], secreted Ly-6/uPAR-related protein 1 [SLURP1], serum amyloid A, fibrinogen alpha chain and pancreatic prohormone) were confirmed to be removed by the dialysis procedure using targeted selected-reaction monitoring MS/MS. Furthermore, capture of HNP-1 and SLURP1 was also validated by Western blot. Interestingly, further analyses of SLURP1 in serum indicated that this protein was 3-fold higher in cholestatic patients than in controls. Proteins captured by MARS share certain structural and biological characteristics, and some of them have important biological functions. Therefore, their removal could be related either to therapeutic or possible adverse effects associated with albumin dialysis.

Objective: Proteases are considered critical in peri-cystic tissue degradation required in jaw cyst expansion. We studied the expression of the plasminogen activation system (plasminogen activators; their inhibitor type-1, PAI-1; the receptor for the urokinase-type plasminogen activator, uPAR) in follicular and inflammatory cysts of the jaw, to identify a possible role of this system in jaw cyst enlargement., Materials and Methods: Jaw cysts were collected by therapeutic enucleation. ELISA and casein zymography were used to measure and characterize plasminogen activators in cyst fluid. By immunohistochemistry we examined the presence of uPAR in cyst walls and inflammatory cells, and by Western blotting the molecular forms of uPAR within the cyst fluid., Results: Inflammatory cysts fluid contained higher amounts of plasminogen activators of the urinary-type (uPA), and lower amounts of PAI-1, when compared to follicular cysts fluid. Epithelial layers of both types of cysts and inflammatory cells expressed uPAR. Native 3-domain uPAR was scarcely detectable within cysts, where its cleavage was accounted for by uPA., Conclusion: These data suggest a plasminogen activation-dependent mechanism of cyst enlargement, where only the outward uPAR expressed on epithelial cells and on inflammatory cells direct the peri-cystic protease cascade, in a way similar to tumor enlargement within tissues., (© 2010 John Wiley & Sons A/S.)

The prognostic and/or predictive value of the cancer biomarkers, urokinase-type plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor [PAI]-1), determined by ELISA in tumor-tissue extracts, was demonstrated for several cancer types in numerous clinically relevant retrospective or prospective studies, including a multicenter breast cancer therapy trial (Chemo-N0). Consequently, for the first time ever for any cancer biomarker for breast cancer, uPA and PAI-1 have reached the highest level of evidence, level-of-evidence-1. At present, two other breast cancer therapy trials, NNBC-3 and Plan B, also incorporating uPA and PAI-1 as treatment-assignment tools are in effect. Furthermore, small synthetic molecules targeting uPA are currently in Phase II clinical trials in patients afflicted with advanced cancer of the ovary, breast or pancreas.

In cancer, the serine protease urokinase-type plasminogen activator, its inhibitor (plasminogen activator inhibitor type-1) and the receptor (CD87), among other proteolytic factors, are involved in tumor cell dissemination and turnover of the extracellular matrix. Unsurprisingly, a battery of very uniform data, amassed since the end of the 1990s, has put these members of the plasminogen activation system into the forefront of prognostic/predictive cancer biomarkers relevant to predict the clinical course of cancer patients and their response to cancer therapy. The present review focuses on the molecular characteristics of the disease forecast biomarkers urokinase-type plasminogen activator and plasminogen activator inhibitor type-1, and techniques to quantitatively assess these cancer biomarkers, in the context of potential clinical application and personalized disease management.