Helle Pappot

The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl‐phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme‐linked immunosorbent assay (ELISA) has been applied for quantttation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma‐uPAR value ±SD was 31 ± 15 pm, median 28 (range 11‐108), and the corresponding value for six PNH patients was 116±67 pm, median 90 (range 61‐228). The elevated uPAR‐level in PNH patients was highly significant (Mann‐Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro‐uPA by uPAR in plasma may interfere with the appropriate binding of pro‐uPA to cell‐bound uPAR and therefore inhibit cell‐associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR‐mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.

Association between plasma concentrations of plasminogen activator inhibitor-1 and survival in patients with colorectal cancer

Editorial by Verspaget Invasion by cancer cells requires proteases such as the serine protease plasmin to degrade the cellular matrix. Plasmin is formed from its zymogen, plasminogen, a reaction catalysed by urokinase type plasminogen activator—which is implicated in invasion1—and partly regulated by plasminogen activator inhibitors. The active form of the inhibitor complexes with free and receptor bound active urokinase plasminogen activator and is bound by vitronectin in plasma and extracellular matrix.2 A high concentration of plasminogen activator inhibitor-1 in biopsy specimens from tumours is associated with a poor prognosis,3 and some patients with ovarian cancer have raised plasma concentrations of plasminogen activator inhibitor-1.4 We studied the prognostic importance of plasma concentrations of plasminogen activator inhibitor-1 in patients with colorectal …

Elevated plasma levels of urokinase plasminogen activator receptor in non-small cell lung cancer patients

The urokinase plasminogen activator (uPA) is involved in extracellular matrix degradation during cancer invasion. Binding of uPA to a specific cell surface receptor (uPAR) is a key step in this process. We have previously reported that high levels of uPAR in squamous cell lung cancer tissue extracts are associated with poor prognosis (Pedersen et al., Cancer Res 1994, 54, 4671-4675). Recently we found that uPAR is present in blood plasma from healthy donors as determined by enzyme-linked immunosorbent assay (ELISA) and chemical cross-linking. We now report that uPAR in plasma from 17 patients with non-small cell lung cancer (NSCLC) was significantly higher than in 30 healthy controls (P = 0.0004), while no significant increase was found in plasma from 14 patients with small cell lung cancer (SCLC). The increased levels of uPAR in the plasma from NSCLC patients is likely to be due to release of uPAR from the tumour tissue, and may, therefore, be related to prognosis.

Vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 expression in non-small cell lung cancer patients: Relation to prognosis

BACKGROUND The majority of patients with non-small cell lung cancer (NSCLC) are diagnosed with advanced inoperable disease. While treatment with conventional chemotherapy has improved during the last decade the 5 years survival is still modest. Novel drugs, which selectively target aberrant elements in neoplastic cells and their microenvironment have recently been and are continuously developed including drugs inhibiting the angiogenic system. Angiogenic factor vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) seem to play key roles in tumour-induced angiogenesis. Previous studies have been inconclusive on the topic of a role for VEGF and its receptor as prognostic factors in NSCLC. METHODS Paraffin-embedded histological material from 102 patients operated for NSCLC was included and a representative block with lung cancer tissue was selected from each patient for immunohistochemical studies. The sections were incubated with primary monoclonal antibodies to VEGF-A and VEGFR2. The expression of the immunohistochemical staining was assessed semi-quantitatively by estimating the percentage and the intensity of tumour cells stained on whole tumour slides. Kaplan-Meier survival curves were generated to evaluate the significance of immunohistochemical VEGF-A and VEGFR2 expression for the prognosis. RESULTS VEGF-A and VEGFR2 expression was observed in the majority of NSCLC patients. VEGF-A expression showed a correlation to histological type with increased expression in adenocarcinomas as compared to squamous cell carcinomas. There was no statistically significant correlation between VEGF-A and VEGFR2 expression and age, gender or stage at diagnosis. Finally there was no relation between expression of VEGF-A and VEGFR2, nor an effect of high expression of both VEGF-A and VEGFR2 on survival. CONCLUSION In conclusion VEGF-A and VEGFR2 are expressed in NSCLC, but the immunohistochemical expression of VEGF-A and VEGFR2 has no prognostic impact in NSCLC. We show that the histological subgroups of NSCLC express VEGF-A differently, with adenocarcinomas having the highest amount. Whether these markers might be useful as clinically reliable predictive markers remains to be solved.

Prognostic and predictive value of intact and cleaved forms of the urokinase plasminogen activator receptor in metastatic prostate cancer

The purpose of this study was to investigate the prognostic value of different forms of the urokinase receptor, uPAR, in serum from prostate cancer (PC) patients.

The liberated domain I of urokinase plasminogen activator receptor--a new tumour marker in small cell lung cancer.

The prognosis of small cell lung cancer (SCLC) remains poor with a 5‐year survival rate of 4–6%. In non‐small cell lung cancer (NSCLC), high levels of intact and cleaved forms of the receptor for urokinase plasminogen activator (uPAR) are significantly associated with short overall survival. Our aim was therefore to determine the prognostic value of the different uPAR forms in blood from SCLC patients. Serum samples from 92 treatment naive SCLC patients were analysed. Intact uPAR, uPAR(I–III), intact and cleaved uPAR, uPAR(I–III) + uPAR(II–III) and the liberated domain I, uPAR(I) were measured using time‐resolved fluorescence immunoassays (TR‐FIA 1–3). Assessment of association of the uPAR forms to overall survival (OS) was done using Cox regression analysis adjusted for clinical covariates [age, gender, stage, lactate dehydrogenase (LDH), WHO performance status (PS)]. Multivariate survival analysis demonstrated that high levels of uPAR(I) were significantly (p = 0.009) associated with short overall survival (OS). Patients with uPAR(I) levels above the second tertile had a hazard ratio (HR) of 1.9 (95% confidence interval (CI): 1.1–3.3), compared to patients with levels below the first tertile. High serum uPAR(I) levels are associated with short OS in SCLC patient, independent of LDH and PS.

Prognostic significance of urokinase plasminogen activator receptor and its cleaved forms in blood from patients with non‐small cell lung cancer

Urokinase plasminogen activator (uPA) cleaves its three‐domain cell surface receptor, uPAR, liberating domain I [uPAR(I)] and leaving the cleaved uPAR(II‐III) on the cell surface. Both intact and cleaved uPAR can be shed from the cell surface. uPAR(I) was previously shown to be a prognostic factor in lung tumour extracts. Here we analyse uPAR forms in blood from patients with non‐small cell lung cancer (NSCLC). Preoperatively sampled plasma/serum from 32 patients with NSCLC was analysed. Three time‐resolved fluoroimmunoassays (TR‐FIAs) measuring intact uPAR(I‐III) (TR‐FIA 1), uPAR(I‐III) + uPAR(II‐III) (TR‐FIA 2) and uPAR(I) (TR‐FIA 3) were applied. The Spearman rank correlations between plasma and serum levels of uPAR(I‐III), uPAR(I‐III) + uPAR(II‐III), and uPAR(I) were 0.89, 0.94 and 0.68 respectively. Survival analysis demonstrated that high levels of all uPAR forms were associated with shorter survival. Adjusted for histological subtype high plasma uPAR(I‐III) and uPAR(I) levels as well as serum uPAR(I) levels were significantly associated with shorter OS (hazards ratios = 4.3, 2.8 and 3.8 respectively). High blood levels of intact uPAR and its cleaved forms are associated with poor prognosis in NSCLC.

Urokinase receptor forms in serum from non-small cell lung cancer patients: Relation to prognosis

OBJECTIVES To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients. MATERIALS AND METHODS Serum sampled preoperatively was available from 171 patients radically resected for NSCLC. Intact uPAR, uPAR(I-III), intact and cleaved uPAR, uPAR(I-III)+uPAR(II-III) and the liberated uPAR(I) were measured by time-resolved fluorescence immunoassays (TR-FIAs 1-3). RESULTS High serum levels of each of the three uPAR forms were associated with short overall survival (OS). In a multivariate survival analysis uPAR(I-III) (hazard ratio (HR)=2.3, 95% confidence interval (CI): 1.2-4.5, p=0.015) and uPAR(I) (hazard ratio (HR)=1.5, 95% CI: 1.0-2.2, p=0.0497) remained significant prognostic parameters independent of stage, histology, age, performance status and therapy. CONCLUSIONS This retrospective study shows that uPAR(I-III) and uPAR(I) in serum are independent prognostic factors in patients radically operated for NSCLC. Further prospective studies are needed to validate these markers for clinical use.

First-in-human uPAR PET: Imaging of Cancer Aggressiveness

A first-in-human clinical trial with Positron Emission Tomography (PET) imaging of the urokinase-type plasminogen activator receptor (uPAR) in patients with breast, prostate and bladder cancer, is described. uPAR is expressed in many types of human cancers and the expression is predictive of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with 64Cu for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of 64Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment with laboratory blood screening tests was performed before and after PET ligand injection. In a subgroup of the patients, the in vivo stability of our targeted PET ligand was determined in collected blood and urine. No adverse or clinically detectable side effects in any of the 10 patients were found. The ligand exhibited good in vivo stability and fast clearance from plasma and tissue compartments by renal excretion. In addition, high uptake in both primary tumor lesions and lymph node metastases was seen and paralleled high uPAR expression in excised tumor tissue. Overall, this first-in-human study therefore provides promising evidence for safe use of 64Cu-DOTA-AE105 for uPAR PET imaging in cancer patients.

ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

A sandwich‐type ELISA has been developed for the assessment of complexes between urokinase‐type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti‐uPA antibodies and a biotinylated mouse anti‐uPAR monoclonal antibody (MAb). The detection limit of the assay is approximately 0.5 fmol/ml. A linear dose‐response is obtained with up to 40 fmol/ml of uPA:uPAR complexes, while uPA and uPAR separately do not cause any response in the ELISA. A buffer which has been used previously for optimal extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti‐uPA or anti‐uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross‐linked uPA:uPAR complexes added to tumor extracts varies between 80% and 105%. The intra‐ and inter‐assay variation coefficients are 5.3% and 9.8%, respectively. Furthermore, a peptide antagonist for uPAR was employed to evaluate de novo uPA:uPAR complex formation during tumor tissue extraction and the immunoassay procedure. Our results strongly indicate that de novo complex formation is a major factor to consider and that complexes analyzed in the presence of this antagonist represent original uPA:uPAR complexes present prior to tumor tissue processing. The present ELISA appears suitable for studying the potential prognostic impact of uPA:uPAR complexes in lung tumor tissue as well as other types of cancer. Int. J. Cancer 72:416–423, 1997.