High PAI-1 Research Articles

Antithrombin Deficiency Is Associated with Prothrombotic Plasma Fibrin Clot Phenotype.

Deficiency of antithrombin increases risk of venous thromboembolism. We hypothesized that antithrombin deficiency affects fibrin clot structure and function. We evaluated 148 patients (age: 38 [32-50] years; 70% women) with genetically confirmed antithrombin deficiency and 50 healthy controls. Fibrin clot permeability (Ks) and clot lysis time (CLT) along with thrombin generation capacity were assessed before and after antithrombin activity normalization in vitro. Antithrombin-deficient patients had lower antithrombin activity (-39%) and antigen levels (-23%) compared with controls (both p < 0.01). Prothrombin fragment 1 + 2 levels were 26.5% higher in patients with antithrombin deficiency than in controls along with 94% increased endogenous thrombin potential (ETP) and 108% higher peak thrombin (all p < 0.01). Antithrombin deficiency was associated with 18% reduced Ks and 35% prolonged CLT (both p < 0.001). Patients with type I (n = 65; 43.9%) compared with type II antithrombin deficiency (n = 83; 56.1%) had 22.5% lower antithrombin activity (p < 0.001) and despite similar fibrinogen levels, 8.4% reduced Ks, 18% prolonged CLT, and 30% higher ETP (all p < 0.01). Reduced Ks was associated with lower antithrombin antigen level (β = - 6.1, 95% confidence interval [CI]: -1.7 to -10.5), while prolonged CLT was associated with lower antithrombin antigen (β = - 69.6, 95% CI: -9.6 to -129.7), activity (β = - 2.4, 95% CI: -0.3 to -4.5), higher PAI-1 (β = 12.1, 95% CI: 7.7-16.5), and thrombin-activatable fibrinolysis inhibitor levels (β = 3.8, 95% CI: 1.9-5.7). Addition of exogenous antithrombin reduced ETP (-42%) and peak thrombin (-21%), and improved Ks (+8%) and CLT (-12%; all p < 0.01). Our study suggests that enhanced thrombin generation and prothrombotic plasma fibrin clot phenotype can contribute to increased risk of thrombosis in patients with antithrombin deficiency.

Anti-PAI-1 Monoclonal Antibody Inhibits the Metastasis and Growth of Esophageal Squamous Cell Carcinoma.

Plasminogen activator inhibitor (PAI-1) is highly expressed in esophageal squamous cell carcinoma (ESCC) and strongly contributes to metastasis, making it a potential target for ESCC therapy. However, the antibodies and inhibitors targeting PAI-1 have not shown good therapeutic effect in the in vivo experiments yet. Here, we generated a panel of novel monoclonal antibodies (mAbs) against PAI-1. Analysis of PAI-1 expression in 90 tissue specimens and 128 serum specimens from ESCC patients with these mAbs confirmed that PAI-1 levels was significantly correlated with metastasis and poor survival. In addition, we found that high PAI-1 expression contributed to the enhanced motility and invasiveness of two ESCC cell lines. Next, mAb-1E2 and mAb-2E3, which have highest affinity with PAI-1, were shown to possess strong inhibitory effects on ESCC migration and invasion. Anti-tumor and anti-metastatic effects of mAb-2E3 were further demonstrated in the experimental animal models. Finally, LRP1 was identified as key factor mediating the pro-invasive function of PAI-1 and the anti-invasive capacity of mAb-2E3 in ESCC cells. The mAb-2E3 markedly decreased STAT1 phosphorylation levels and blocked the binding between PAI-1 and LRP1-ClusterII domain. Collectively, mAb-2E3 developed by our lab may be an effective antibody drug which can be used for anti-metastatic therapy in ESCC.

Plasminogen Activator Inhibitor-1 in Acute Myeloid Leukemia: Is It Useful in Predicting Response to Induction Remission Therapy?

BACKGROUND: Coagulation and fibrinolytic abnormalities are common in patients with acute myeloblastic leukemia (AML). Plasminogen activator inhibitor (PAI) activity is believed to be elevated during the initial diagnosis and relapse of AML patients. AIM: This study aimed to evaluate the levels of plasma PAI-1 activity in AML patients before and after remission induction. METHODS: Thirty AML patients and 20 healthy volunteers were included in this study. The patients were classified according to AML-FAB subtypes. All patients received 7+3 induction chemotherapy. They were evaluated for complete remission after induction chemotherapy and followed up for 6 months. RESULTS: PAI-1 activity was measured by ELISA immunoassay. PAI-1 activity was significantly higher in AML patients than the control group (P=0.016), whereas there was insignificant difference among patients in various AML subgroups (P&gt;0.05). Before and after treatment, there was a significant difference in PAI-1 activity between patients with active disease and those at remission (P= 0.023 and &lt;0.0001, respectively). CONCLUSION: High PAI-1 activity in AML patients is found to be associated with poor response to treatment.

Low Transforming Growth Factor-β Pathway Activity in Cervical Adenocarcinomas.

Cervical cancer is the fourth most common cancer in women worldwide. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the most common histological types, with AC patients having worse prognosis. Over the last two decades, incidence rates of AC have increased, highlighting the importance of further understanding AC tumorigenesis, and the need to investigate new treatment options. The cytokine TGF-β functions as a tumour suppressor in healthy tissue. However, in tumour cells this suppressive function can be overcome. Therefore there is an increasing interest in using TGF-β inhibitors in the treatment of cancer. Here, we hypothesize that TGF-β plays a different role in SCC and AC. Analysis of RNA-seq data from the TCGA, using a TGF-β response signature, resulted in separate clustering of the two subtypes. We further investigated the expression of TGF-β-signalling related proteins (TβR1/2, SMAD4, pSMAD2, PAI-1, αvβ6 and MMP2/9) in a cohort of 62 AC patients. Low TβR2 and SMAD4 expression was associated with worse survival in AC patients and interestingly, high PAI-1 and αvβ6 expression was also correlated with worse survival. Similar correlations of TβR2, PAI-1 and αvβ6 with clinical parameters were found in previously reported SCC analyses. However, when comparing expression levels between SCC and AC patient samples, pSMAD2, SMAD4, PAI-1 and αvβ6 showed lower expression in AC compared to SCC. Because of the low expression of core TβR1/2, (p-)SMAD2 and SMAD4 proteins and the correlation with worse prognosis, TGF-β pathway most likely leads to tumour inhibitory effects in AC and therefore the use of TGF-β inhibitors would not be recommended. However, given the correlation of PAI-1 and αvβ6 with poor prognosis, the use of TGF- β inhibitors might be of interest in SCC and in the subsets of AC patients with high expression of these TGF-β associated proteins.

Age-Related Increase of Collagen/Fibrin Deposition and High PAI-1 Production in Human Nasal Polyps.

Objective: Our previous studies showed an age-related increased prevalence of nasal polyps (NP) and reduced production of S100A8/9 in elderly patients with chronic rhinosinusitis with NP (CRSwNP). In this study, we investigated an unbiased age-related gene expression profile in CRSwNP subjects and healthy controls, and further identified the differences in their tissue remodeling. Methods: Microarrays using NP and uncinate tissues from health controls (elderly, age ≥65 vs. non-elderly, age 18–49) were performed, and differentially regulated genes were analyzed. Quantitative real-time PCR (qPCR), Immunostaining, Periodic acid-Schiff (PAS), trichrome staining, Western blot, and ELISA were performed for further investigation. Results: Microarrays identified differentially expressed genes according to disease and age; 278 in NP vs. controls, 75 in non-elderly NP vs. non-elderly controls, and 32 in elderly NP vs. elderly controls. qPCR confirmed that the PLAT gene was downregulated and the SERPINB2 gene upregulated in NP vs. controls. The serous glandular cell-derived antimicrobial protein/peptide-related genes such as BPIFB3, BPIFB2, LPO, and MUC7 were remarkably reduced in NP, regardless of age. SERPINE1 gene (plasminogen activator inhibitor-1, PAI-1) expression was significantly increased in elderly NP versus elderly controls. IHC and western blot confirmed significantly decreased production of MUC7 and LPO in NP versus controls. There was a trend of age-related reduction of submucosal gland cells in normal controls. Trichrome and immunofluorescence staining demonstrated an age-related increase of collagen and fibrin deposition in NP, consistent with increased PAI-1 production. Conclusion: This study demonstrated age-related differential glandular remodeling patterns and fibrosis in NP and normal controls. PAI-1 expression was significantly increased in elderly NP versus elderly controls, suggesting PAI-1 as a potential treatment target in elderly NP.

Comorbidity-associated glutamine deficiency is a predisposition to severe COVID-19.

SARS-CoV-2 vaccinations have greatly reduced COVID-19 cases, but we must continue to develop our understanding of the nature of the disease and its effects on human immunity. Previously, we suggested that a dysregulated STAT3 pathway following SARS-Co-2 infection ultimately leads to PAI-1 activation and cascades of pathologies. The major COVID-19-associated metabolic risks (old age, hypertension, cardiovascular diseases, diabetes, and obesity) share high PAI-1 levels and could predispose certain groups to severe COVID-19 complications. In this review article, we describe the common metabolic profile that is shared between all of these high-risk groups and COVID-19. This profile not only involves high levels of PAI-1 and STAT3 as previously described, but also includes low levels of glutamine and NAD+, coupled with overproduction of hyaluronan (HA). SARS-CoV-2 infection exacerbates this metabolic imbalance and predisposes these patients to the severe pathophysiologies of COVID-19, including the involvement of NETs (neutrophil extracellular traps) and HA overproduction in the lung. While hyperinflammation due to proinflammatory cytokine overproduction has been frequently documented, it is recently recognized that the immune response is markedly suppressed in some cases by the expansion and activity of MDSCs (myeloid-derived suppressor cells) and FoxP3+ Tregs (regulatory T cells). The metabolomics profiles of severe COVID-19 patients and patients with advanced cancer are similar, and in high-risk patients, SARS-CoV-2 infection leads to aberrant STAT3 activation, which promotes a cancer-like metabolism. We propose that glutamine deficiency and overproduced HA is the central metabolic characteristic of COVID-19 and its high-risk groups. We suggest the usage of glutamine supplementation and the repurposing of cancer drugs to prevent the development of severe COVID-19 pneumonia.

Oleuropein is a natural inhibitor of PAI-1-mediated proliferation in human ER-/PR- breast cancer cells.

Elevated expression of PAI-1 has been widely linked with adverse outcomes in a variety of human cancers, such as breast, gastric and ovarian cancers, rendering PAI-1 a prognostic biomarker. As a result, several chemical inhibitors are currently being developed against PAI-1; however, the clinical setting where they might confer survival benefits has not yet been elucidated. RNA sequencing data analysis from the TCGA/GTEx cancer portals (n = 3607 samples). In silico molecular docking analyses to predict functional macromolecule interactions. ER-/PR- (MDA-MB-231) and ER+/PR+ (MCF-7) breast cancer cell lines implemented to assess the effect of oleuropein as a natural inhibitor of PAI-1-mediated oncogenic proliferation. We show that high PAI-1 levels inversely correlate with ER and PR expressions in a wide panel of estrogen/progesterone-responsive human malignancies. By implementing an in silico molecular docking analysis, we identify oleuropein, a phenolic component of olive oil, as a potent PAI-1-binding molecule displaying increased affinity compared to the other olive oil constituents. We demonstrate that EVOO or oleuropein treatment alone may act as a natural PAI-1 inhibitor by incrementally destabilising PAI-1 levels selectively in ER-/PR- breast cancer cells, accompanied by downstream caspase activation and cell growth inhibition. In contrast, ER+/PR+ breast cancer cells, where PAI-1 expression is absent or low, do not adequately respond to treatment. Our study demonstrates an inverse correlation between PAI-1 and ESR1/PGR levels, as well as overall patient survival in estrogen/progesterone-responsive human tumours. With a focus on breast cancer, our data identify oleuropein as a natural PAI-1 inhibitor and suggest that oleuropein-mediated PAI-1 destabilisation may confer clinical benefit only in ER-/PR- tumours.

Tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in patients with symptomatic lower extremity artery disease.

Background Lower extremity artery disease (LEAD) involves progressive arterial narrowing manifested by intermittent claudication (IC). LEAD entails endothelial dysfunction and fibrinolytic disorders. In the current study, we analyze the selected parameters of the fibrinolytic system in the blood of patients with symptomatic LEAD depending on clinical parameters. Methods The test group was comprised of 80 patients with diagnosis of LEAD based on Ankle-Brachial Index (ABI) test (27 female/53 male) with an average age of 63.5±9 years. The control group included 30 healthy, non-smoking volunteers (10 female/20 male), with the median age of 56±6 years. The research material - venous blood - was sampled to determine the concentrations of tissue-type plasminogen activator (t-PA Ag), plasminogen activator inhibitor type 1 (PAI-1 Ag), D-dimer, fibrinogen, and platelet count (PLT). Results We found elevated concentrations of t-PA Ag, PAI-1 Ag, D-dimer, and fibrinogen in the plasma of subjects with symptomatic LEAD. Various stages of the Fontaine classification demonstrated a gradual, statistically significant increase in the concentrations of fibrinogen and PLT count as the disease progressed. More so, in the subgroup of LEAD patients aged ≥65 years, we observed significantly higher levels of D-dimer than in the group of younger subjects. In addition to that, the LEAD group demonstrated negative correlations of IC distance, fibrinogen concentrations, and PLT count, negative correlations of ABI at rest and concentrations of D-dimer and PLT count, as well as positive correlations between age and D-dimer levels. Conclusions High t-PA Ag concentrations in LEAD patients suggest damage to the endothelium which comprises the main source of this factor. With high PAI-1 Ag levels, inactive fibrinolytic t-PA-PAI-1 complexes are formed. Increasing fibrinogen concentrations at the subsequent stages in accordance with the Fontaine classification, indicate increasing inflammation. Moreover, heightened values of D-dimer reflect an increased secondary fibrinolysis activation as patients get older and impaired extremity vascularization, manifested by the decreasing ABI, progresses.

Tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in patients with symptomatic lower extremity artery disease.

Lower extremity artery disease (LEAD) involves progressive arterial narrowing manifested by intermittent claudication (IC). LEAD entails endothelial dysfunction and fibrinolytic disorders. In the current study, we analyze the selected parameters of the fibrinolytic system in the blood of patients with symptomatic LEAD depending on clinical parameters. The test group was comprised of 80 patients with diagnosis of LEAD based on Ankle-Brachial Index (ABI) test (27 female/53 male) with an average age of 63.5±9 years. The control group included 30 healthy, non-smoking volunteers (10 female/20 male), with the median age of 56±6 years. The research material - venous blood - was sampled to determine the concentrations of tissue-type plasminogen activator (t-PA Ag), plasminogen activator inhibitor type 1 (PAI-1 Ag), D-dimer, fibrinogen, and platelet count (PLT). We found elevated concentrations of t-PA Ag, PAI-1 Ag, D-dimer, and fibrinogen in the plasma of subjects with symptomatic LEAD. Various stages of the Fontaine classification demonstrated a gradual, statistically significant increase in the concentrations of fibrinogen and PLT count as the disease progressed. More so, in the subgroup of LEAD patients aged ≥65 years, we observed significantly higher levels of D-dimer than in the group of younger subjects. In addition to that, the LEAD group demonstrated negative correlations of IC distance, fibrinogen concentrations, and PLT count, negative correlations of ABI at rest and concentrations of D-dimer and PLT count, as well as positive correlations between age and D-dimer levels. High t-PA Ag concentrations in LEAD patients suggest damage to the endothelium which comprises the main source of this factor. With high PAI-1 Ag levels, inactive fibrinolytic t-PA-PAI-1 complexes are formed. Increasing fibrinogen concentrations at the subsequent stages in accordance with the Fontaine classification, indicate increasing inflammation. Moreover, heightened values of D-dimer reflect an increased secondary fibrinolysis activation as patients get older and impaired extremity vascularization, manifested by the decreasing ABI, progresses.

Assessment of plasminogen activator inhibitor-1 in obese Egyptian children

BackgroundPlasminogen activator inhibitor-1 (PAI-1) is mainly produced in the liver and in the adipose tissue. Normal fibrin clearance mechanisms were found to be affected by high plasma PAI-1 levels and thus increases risk of thrombosis. The aim of the current study was to expound the childhood obesity effect on circulating PAI-1 and interpret the relation of PAI-1 to metabolic syndrome. This cross-sectional study was conducted on 43 obese children following in the Children Hospital and compared to 44 healthy sex- and age-matched controls. All recruited cohort are subjected to anthropometric measurements: weight, height, BMI, waist circumference, hip circumference, and skin fold thickness (biceps, triceps, and subscapular), and laboratory investigations in the form of lipid profile, fasting blood sugar, fasting insulin, insulin resistance estimated by HOMA-IR, and plasminogen activator inhibitor-1.ResultsThe level of plasminogen activator inhibitor-1 in the obese group was significantly higher than that in the control group (47.98 ± 17.42 vs. 28.00 ± 11.35 respectively). PAI-1 showed positive significant correlation to anthropometric measurements: BMI (p = 0.000), weight (p = 0.000), biceps skin fold thickness (p = 0.04), triceps skin fold thickness (p = 0.4), and subscapular skin fold thickness (p = 0.04). Also, a significant positive correlation was found between PAI-1 and systolic (p = 0.000) and diastolic blood pressure (p = 0.04). Positive correlations were found between PAI-1 and cholesterol (p = 0.000), triglycerides (p = 0.02), LDL-c (p = 0.000), insulin (p = 0.000), and HOMA-IR (r = 0.5, p = 0.02).ConclusionFat mass accumulation is related to high PAI-1 levels, which might in turn contribute to cardiovascular risk. Plasminogen Activator Inhibitor-1 is a good predictive test for metabolic syndrome in obese children.

Abstract 4930: PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC

Abstract Lung cancer management has been transformed by the discovery and effective targeting of driver mutations such as epidermal growth factor receptor (EGFR). Tyrosine Kinase Inhibitors (TKIs) that block EGFR kinase activity such as erlotinib and osimertinib cause dramatic tumor shrinkage and prolonged responses in patients whose tumors have activating EGFR mutations. However, resistance universally develops. Drug persister cells with properties of cancer stem cell-like cells play a major role in drug resistance. In tumors without pre-existing resistant clones, some tumor clonogens survive, despite expressing the driver mutation, to acquire target reactivation or bypass resistance mechanisms. Identification of biomarkers that are mechanistically linked to EGFR TKI resistant cell populations would have clinical utility in early determination of disease recurrence in EGFR mutant NSCLC patients. Previously, we have reported that EGFR-TKI treatment activates Notch3, and here we have identified that Notch3 activates β-catenin transcriptional target, PAI1, which could play a role in drug persistence and could be used for early identification of disease recurrence. Our in vitro ELISA studies have identified an increase in secreted PAI1 levels after treatment with the EGFR TKIs erlotinib and osimertinib demonstrate that measuring PAI1 levels could serve as an indicator of EGFR TKI persistence. To further validate PAI1 as a surrogate biomarker for EGFR TKI treatment, we performed a retrospective study in NSCLC patients (n = 42) with EGFR mutant tumors. Using ELISA to detect PAI1 protein in pre- and post-treatment serum samples as a mechanism-based indicator for β-catenin activation, we observed a broad range in pre-treatment PAI1 levels, spanning from 0.8 ng/ml to 134 ng/ml, suggesting a variable baseline β-catenin activation. Doing survival analysis after dichotomizing patients into low and high baseline PAI1 level groups, we found patients with low basal PAI1 levels had a significantly shorter PFS with EGFR TKI treatment compared to patients with high basal levels. Comparing patients with a ≥ 2-fold induction to those with less than 2-fold, there was a similar trend for worsened survival in those who showed significant induction of PAI1. Overall, we demonstrate that PAI1 expression is a candidate marker for β-catenin activation and the development of drug persisters; thus, it is a potential surrogate marker for poor outcome in patients receiving EGFR TKI therapy and a potential biomarker for selecting patients for β-catenin-targeted therapy. Citation Format: Rajeswara Rao Arasada, Walter Wang, Shankar Suman, David Paul Carbone. PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4930.

The Effect of PAI-1 Gene Variants and PAI-1 Plasma Levels on Development of Thrombophilia in Patients With Klinefelter Syndrome.

Klinefelter syndrome (KS) is a common sex chromosome-related abnormality seen among men. KS negatively affects spermatogenesis and testosterone production. It increases the risk of thrombosis but its molecular mechanism has not been well described yet. Elevated PAI-1 is a risk factor for thrombosis. The rs1799889 polymorphism located in the promoter region of the PAI-1 gene was detected in patients with deep venous thrombosis. In this study, the PAI-1 gene variant and its plasma levels in KS patients were examined. Forty-one KS patients (47, XXY) and 50 age-matched healthy controls participated. DNA was isolated from peripheral blood and a real-time PCR method was used to detect known SNPs in the PAI-1 gene. In addition, PAI-1 plasma levels were measured by using ELISA method. There was no significant difference between PAI-1 gene polymorphisms of KS patients and controls (p > .05). The significant difference was observed in PAI-1 plasma levels between two groups (high PAI-1 plasma level in KS patients compared to controls). The patients’ group mean was 55.13 and control group mean in PAI-1 level was 29.89 ng/ml (p = .020). Clinical features related to thromboembolism especially varicose veins were detected in KS patients frequently (p = .04). These results suggest that thromboembolism related to clinical features is seen more frequently in cases with KS, but it may not be dependent only on the PAI-1 gene polymorphism structure.

Clinical Investigation of Coagulation Markers for Early Detection of Sepsis-Induced Disseminated Intravascular Coagulation: A Single-Center, Prospective Observational Study.

Disseminated intravascular coagulation (DIC) often complicates sepsis, and its early treatment is crucial for improving patient outcomes. Coagulation markers may enable earlier diagnosis of DIC. The purpose of this study was to evaluate whether the risk of DIC onset can be predicted using coagulation markers. Patients who showed symptoms of systemic inflammatory response syndrome ≥2 and the quick Sequential Organ Failure Assessment score ≥2 points were investigated. All blood samples collected from the time of hospital admission to 7 days postadmission were investigated. Patients were classified according to time of DIC onset (1) no DIC group (not DIC developed), (2) pre-DIC group (DIC onset >24 hours after admission), (3) DIC group (DIC onset at time of the admission) and according to cutoff values of coagulation markers, High group and Low group. Statistical differences were analyzed by log-rank test, Kruskal-Wallis rank test, and Friedman test. A total of 107 patients were enrolled in the study. Soluble fibrin (SF), plasminogen activator inhibitor (PAI)-1, and d-dimer levels were significantly increased even under pre-DIC conditions. Japanese Association for Acute Medicine (JAAM) DIC scores increased significantly over time in the High SF group (≥31.0 µg/mL) and High PAI-1 group (≥49.0 ng/mL), while JAAM DIC scores in the Low SF group remained ≤3 until day 7. We proposed the cutoff values of SF as 31 µg/mL to detect early phase of DIC. Soluble fibrin might be useful not only to predict DIC but also to exclude a diagnosis of DIC.

Prognostic implications of the co-detection of the urokinase plasminogen activator system and osteopontin in patients with non-small-cell lung cancer undergoing radiotherapy and correlation with gross tumor volume.

The urokinase plasminogen activator system (uPA, uPAR, PAI‑1) is upregulated in cancer and high plasma levels are associated with poor prognosis. Their interaction with hypoxia-related osteopontin (OPN) which is also overexpressed in malignant tumors suggests potential clinical relevance. However, the prognostic role of the uPA system in the radiotherapy (RT) of non-small-cell lung cancer (NSCLC), particularly in combination with OPN, has not been investigated so far. uPA, uPAR, PAI‑1 and OPN plasma levels of 81patients with locally advanced or metastasized NSCLC were prospectively analyzed by ELISA before RT and were correlated to clinical patient/tumor data and prognosis after RT. uPAR plasma levels were higher in M1; uPA and PAI‑1 levels were higher in M0 NSCLC patients. uPAR correlated with uPA (p < 0.001) which also correlated with PAI‑1 (p < 0.001). The prognostic impact of OPN plasma levels in the RT of NSCLC was previously reported by our group. PAI‑I plasma levels significantly impacted overall (OS) and progression-free survival (PFS). Low PAI‑1 levels were associated with a significantly reduced OS and PFS with a nearly 2‑fold increased risk of death (p = 0.029) and tumor progression (p = 0.029). In multivariate analysis, PAI‑1 levels remained an independent prognostic factor for OS and PFS with a 3‑fold increased risk of death (p = 0.001). If PAI‑1 plasma levels were combined with OPN or tumor volume, we found an additive prognostic impact on OS and PFS with a 2.5- to 3‑fold increased risk of death (p = 0.01). Our results suggest that PAI-1 but not uPA and uPAR might add prognostic information in patients with advanced NSCLC undergoing RT. High pretreatment PAI-1 plasma levels were found predominantly in M0-stage patients and indicate a favorable prognosis as opposed to OPN where high plasma levels are associated with poor survival and metastasis. In combination, PAI-1 and OPN levels successfully predicted outcome and additively correlated with prognosis. These findings support the notion of an antidromic prognostic impact of OPN and PAI-1 plasma levels in the RT of advanced NSCLC.

Prognostic impact of the inclusion of uPA/PAI-1 for adjuvant treatment decision-making in ER+/Her2- pN0 early breast cancers.

Intermediate-risk early breast cancer (EBC) is a heterogeneous group in which adjuvant chemotherapy decision proves to be difficult. Clinical and pathological criteria are sometimes insufficient to determine the best therapeutic options, and validated biomarkers such as uPA/PAI-1, are needed to contribute to the decision-making. The objective of this study was to evaluate the clinical outcome of an unselected ER+/HER2- pN0 EBC cohort of patients in whom the routine clinical decision process included a prospective uPA/PAI-1 determination. This monocentric retrospective study included 520 patients who underwent curative surgery in our institute between 2006 and 2011. Adjuvant therapeutic strategy was decided based on clinical-pathological data, altogether with a routine prospective determination of uPA/PAI-1 tumor levels using fresh, extemporaneously sampled tissue. We evaluated the correlation between uPA/PAI-1 levels, clinical-pathological variables, and the patient's outcome (relapse-free survival, RFS, and overall survival, OS). Median follow-up was 5.4years. The 5- and 10-year RFS rates were ,respectively, 95 and 89%, and the five-year OS rate was 96.3%. Forty percent of tumors had low uPA/PAI-1 levels. Seventy-five percent of patients with low uPA/PAI-1 levels did not receive chemotherapy, when 25% did. Sixty percent of patients with high uPA and/or PAI-1 levels received chemotherapy, while 40% did not. No statistical significant correlation was found between the uPA/PAI-1 levels and RFS or OS. The personalization of the patients' treatment using uPA/PAI-1 tumor levels allows the reversion of the well-known poor prognostic impact of high uPA/PAI-1 levels and strongly supports the use of this biomarker in clinical practice.

Dysfunctional fibrinolysis and cerebral venous thrombosis

Cerebral venous thrombosis (CVT) is an uncommon neurological disease with high morbidity and mortality. Even after extensive thrombophilia screening, majority of the thrombosis cases remain with unknown etiology. Hypofibrinolysis due to acquired or congenital deficiencies or abnormalities in factors in the fibrinolytic cascade is a known cause of thrombosis at any site. In the present study 104 cases of radiologically confirmed CVT cases were investigated for the conventional thrombophilia along with factors in the fibrinolytic cascade to find a possible etiology for the clinical manifestation. Conventional thrombophilia markers which included PC, PS, AT and FVL mutation were detected in 16.3% of the patients. Approximately 19% cases had grossly elevated plasma PAI-1 levels. PAI-1 4G/4G genotype was found to be strongly associated with high PAI-1 levels. 2.9% cases had reduced tPA levels, 1.9% had plasminogen deficiency and 1.9% cases had increased alpha-2-antiplasmin levels. Along with conventional thrombophilia, dysfunctional fibrinolysis is found to be strongly associated with CVT. Understanding the role of risk factors is important for appropriate treatment of this serious disorder.

The urokinase plasminogen activation system in gastroesophageal cancer: A systematic review and meta-analysis.

BackgroundThe urokinase plasminogen activation (uPA) system is a crucial pathway for tumour invasion and establishment of metastasis. Although there is good evidence that uPA system expression is a clinically relevant biomarker in some solid tumours, its role in gastroesophageal cancer is uncertain.ResultsWe identified 22 studies encompassing 1966 patients which fulfilled the inclusion criteria. uPA, uPAR, or PAI-1 expression is significantly associated with high risk clinicopathological features. High uPA expression is associated with a shorter RFS (HR 1.90 95% 1.16–3.11, p = 0.01) and OS (HR 2.21 95% CI 1.74–2.80, p < 0.0001). High uPAR expression is associated with poorer OS (HR 2.21 95%CI 1.82–2.69, p < 0.0001). High PAI-1 expression is associated with shorter RFS (HR 1.96 96% CI 1.07–3.58, p = 0.03) and OS (HR 1.84 95%CI 1.28–2.64, p < 0.0001). There was no significant association between PAI-2 expression and OS (HR 0.97 95%CI 0.48–1.94, p < 0.92) although data was limited.Materials and MethodsWe undertook a systematic review evaluating expression of uPA, urokinase plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1/SerpinE1) and plasminogen activator inhibitor-2 (PAI-2/SerpinB2) on primary oesophageal, gastro-oesophageal junction, and gastric adenocarcinomas. We performed a meta-analysis of clinicopathological associations, overall survival (OS) and recurrence free survival (RFS).ConclusionsWe conclude that the uPA system is a clinically relevant biomarker in primary gastroesophageal cancer, with higher expression of uPA, uPAR and PAI-1 associated with higher risk disease and poorer prognosis. This also highlights the potential utility of the uPA system as a therapeutic target for improved treatment strategies.

Abstract P6-09-28: Prognostic impact of the inclusion of uPA/PAI-1 in ER+/Her2- pN0 early breast cancer adjuvant treatments decision-making

Abstract Purpose: Intermediate-risk early breast cancer (EBC) is a heterogeneous group in which adjuvant chemotherapy decision prove to be difficult. Clinical and pathological criteria might be insufficient to determine the best therapeutic options for patients, and validated biomarkers are urgently needed to help decision making, such as the LOE-I uPA/PAI-1 biomarker. The aim of this study is to evaluate the clinical outcome of a large, unselected, ER+/HER2- pN0 EBC cohort of patients in whom the routine clinical decision process included a prospective uPA/PAI-1 determination. Methods: This monocentric retrospective study included 520 ER+/HER2- pN0/M0 EBC patients who had curative surgery in our center between 2006 and 2011. Adjuvant therapeutic strategy was decided based on clinico-pathological data, altogether with a routine prospective determination of uPA/PAI-1 tumor levels. We evaluated the correlation between uPA/PAI-1 levels, classical clinico-pathological variables and the patient's outcome (relapse free survival, RFS; overall survival, OS). Results: Median age was 54 years (range 27-85). 75% of tumors were classified T1, 25% T2 and above. We found 80.8% of ductal carcinomas, 12.3% of lobular carcinomas and 6.9% of other histological types. SBR grade 1-2 represented 82.5% of our cohort versus 17.5% of grade 3. Mitotic count was &amp;lt;10 in 63.5% of cases, and ≥10 in 36.5%. Peri-tumoral invasion (PVI) was seen in 17% of tumors. Progesterone receptors (PR) were positive in 79.6% of cases. Median follow-up was 5.4 years. 5 and 10 years RFS were respectively 95% and 89% (n=33 local or metastatic relapse). 5 year OS was 96.3%. We found 40% of low uPA/PAI-1 levels, and 60% of high uPA and/or PAI-1 levels. By using uPA/PAI-1 levels in our adjuvant treatment decision-making, 75% of patients with low uPA/PAI-1 levels did not received chemotherapy and 60% of patients with high uPA and/or PAI-1 levels received chemotherapy. 98.3% of our patients received endocrine therapy as adjuvant treatment for at least 5 years. No statistical significant correlation was found between uPA/PAI-1 levels and RFS (p=0.3) or OS (p=0.28). In univariate analysis, tumor size (p=0.048; p=0.0694), histological grade (p=0.007; p=0.0142), PR status (p=0.001; p=0.0002) and mitotic count (p=0.0001; p=0.0001) were statistically correlated with RFS and OS, respectively. Using cox regression model, PR status (p&amp;lt;0.003) and mitotic count (p&amp;lt;0.001) appeared to be strongly correlated with RFS. No changes in significant variables were seen when the analyses were restricted to the grade 2 subgroup (n=339). Conclusion: The individualization of patients' treatment using uPA/PAI-1 tumor levels allows the reversion of the well-known poor prognostic impact of high uPA/PAI-1 levels and strongly support the use of this LOE-I biomarker in clinical practice. PR status and mitotic activity remains independent major prognostic factors in optimally treated patients. The evaluation of the additional impact of multigene signature in this setting needs to be performed. Longer 10-years follow-up needs to be done in this ER+/HER2- subgroup, particularly to evaluate the risk of late relapse after endocrine therapy. Citation Format: Viala M, Alexandre M, Thézenas S, Lamy P-J, Bibeau F, Gutowski M, Colombo P-E, Romieu G, Jacot W, Guiu S. Prognostic impact of the inclusion of uPA/PAI-1 in ER+/Her2- pN0 early breast cancer adjuvant treatments decision-making [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-28.

Inhibition of PAI-1 Limits Tumor Angiogenesis Regardless of Angiogenic Stimuli in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor that secretes various angiogenic factors. The main inhibitor of plasminogen activators, PAI-1 (SERPINE1), has been implicated in tumor progression and angiogenesis, and high PAI-1 expression has been associated with poor prognosis in MPM patients. In this study, we examined the antiangiogenic effects of PAI-1 inhibition in MPM. We administered the PAI-1 inhibitor, SK-216, to orthotopic mouse models in which MPM cells expressing high levels of VEGF (VEGFA) or bFGF (FGF2) were intrapleurally transplanted. SK-216 administration reduced tumor weights and the degree of angiogenesis in intrapleural tumors, irrespective of their angiogenic expression profiles. In addition, a combination of SK-216 and the chemotherapeutic agent cisplatin significantly reduced tumor weights compared with monotherapy, prolonging the survival of animals compared with cisplatin treatment alone. Furthermore, SK-216 inhibited migration and tube formation of cultured human umbilical vein endothelial cells induced by various angiogenic factors known to be secreted by MPM. These findings suggest that PAI-1 inactivation by SK-216 may represent a general strategy for inhibiting angiogenesis, including for the treatment of MPM. Cancer Res; 76(11); 3285-94. ©2016 AACR.

Validation of a modified thromboelastometry approach to detect changes in fibrinolytic activity.

BackgroundThus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans.The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay.MethodsBlood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients.ResultsThirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ng/ml rTPA were in line with the plasma determinants (high PAI-1, high fibrinogen, low tPA activity, and high d-dimers). After cardiothoracic surgery, reduced fibrinogen and platelet levels accounted for the reduced maximum clot firmness. The hypofibrinolytic profile is attributed to tranexamic acid use and elevated PAI-1 levels. The lowest rTPA concentration in cirrhosis resulted in hyperfibrinolysis in only few of the patients. In pregnancy normal profiles were found.DiscussionOur high rTPA concentration demonstrates hypofibrinolytic profiles adequately in sepsis and after cardiothoracic surgery. Our low rTPA concentration of 125 ng/ml seems too high for demonstrating hyperfibrinolysis in cirrhotic liver disease.ConclusionsWe were able to present a validated whole blood ROTEM approach to fibrinolysis testing using added rTPA, which can be of added value next to classical plasma based fibrinolysis assays.