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Despite improved therapy of advanced colorectal cancer, the median overall survival (OS) is still low. A surgical removal has significantly improved survival, if lesions are entirely removed. The purpose of this retrospective explorative study was to evaluate the prognostic value of histological growth patterns (GP) in chemonaive and patients receiving neo-adjuvant therapy. Two-hundred-fifty-four patients who underwent liver resection of colorectal liver metastases between 2007 and 2011 were included in the study. Clinicopathological data and information on neo-adjuvant treatment were retrieved from patient and pathology records. Histological GP were evaluated and related to recurrence free and OS. Kaplan-Meier curves, log-rank test and Cox regression analysis were used. The 5-year OS was 41.8% (95% CI 33.8-49.8%). Growth pattern evaluation of the largest liver metastasis was possible in 224 cases, with the following distribution: desmoplastic 63 patients (28.1%); pushing 77 patients (34.4%); replacement 28 patients (12.5%); mixed 56 patients (25.0%). The Kaplan-Meier analyses demonstrated that patients resected for liver metastases with desmoplastic growth pattern had a longer recurrence free survival (RFS) than patients resected for non-desmoplastic liver metastases (p=0.05). When patients were stratified according to neo-adjuvant treatment in the multivariate Cox regression model, hazard ratios for RFS compared to desmoplastic were: pushing (HR=1.37, 95% CI 0.93-2.02, p=0.116), replacement (HR=2.16, 95% CI 1.29-3.62, p=0.003) and mixed (HR=1.70, 95% CI 1.12-2.59, p=0.013). This was true for chemonaive patients as well as for patients who received neo-adjuvant treatment.

Proteolytic activity and inflammation in the tumour microenvironment affects cancer progression. In colorectal cancer (CRC) liver metastases it has been observed that three different immune profiles are present, as well as proteolytic activity, determined by the expression of urokinase-type plasminogen activator (uPAR).The main objectives of this study were to investigate uPAR expression and the density of macrophages (CD68) and T cells (CD3) as markers of inflammation in resected CRC liver metastases, where patients were neo-adjuvantly treated with chemotherapy with or without the angiogenesis inhibitor bevacizumab. Chemonaive patients served as a control group. The markers were correlated to growth patterns (GP) of liver metastases, i.e. desmoplastic, pushing and replacement GP. It was hypothesised that differences in proteolysis and inflammation could reflect tumour specific growth and therapy related changes in the tumour microenvironment. In chemonaive patients, a significantly higher level of uPAR was observed in desmoplastic liver metastases in comparison to pushing GP (p = 0.01) or replacement GP (p = 0.03). A significantly higher density of CD68 was observed in liver metastases with replacement GP in comparison to those with pushing GP (p = 0.01). In liver metastases from chemo treated patients, CD68 density was significantly higher in desmoplastic GP in comparison to pushing GP (p = 0.03). In chemo and bevacizumab treated patients only a significant lower CD3 expression was observed in liver metastases with a mixed GP than in those with desmoplastic (p = 0.01) or pushing GP (p = 0.05). Expression of uPAR and the density of macrophages at the tumour margin of liver metastasis differ between GP in the untreated patients. A higher density of T cells was observed in the bevacizumab treated patients, when desmoplastic and pushing metastases were compared to liver metastases with a mix of the GP respectively, however no specific correlations between the immun

BACKGROUND: High levels of intact and cleaved forms of the urokinase-type plasminogen activator receptor (uPAR) in both tissue and blood are associated with poor survival in several cancer diseases. The prognostic significance of uPAR in cholangiocarcinoma is unknown. The aims of this study were to determine if pre-treatment serum levels of uPAR forms and a decrease in levels during chemotherapy are predictive of survival in patients with inoperable cholangiocarcinoma.DESIGN AND METHODS: Patients with inoperable cholangiocarcinoma were consecutively included in the training set (n=108). A test set included patients from a different hospital using similar treatment guidelines (n=60). Serum levels of the different uPAR forms were determined using time-resolved fluorescence immunoassays (TR-FIA). The Cox proportional hazards model was used for the uni- and multivariate survival analyses.RESULTS: Baseline level of uPAR(I-III)+uPAR(II-III) was an independent predictor of survival (HR=2.08, 95% CI:1.46-2.97, p<0.0001). Applying the linear predictor from the training set to the test set, it was validated that uPAR(I-III)+uPAR(II-III) predicted overall survival (p=0.049). A high level of uPAR(I-III)+uPAR(II-III) after 2cycles of chemotherapy was associated with poor survival (HR=1.79, 95% CI:1.08-2.97, p=0.023, n=57). This predictor, however, was not significant in the test set (p=0.21, 26 events in 27 patients).CONCLUSION: The baseline level of uPAR(I-III)+uPAR(II-III) is a predictor of survival in inoperable cholangiocarcinoma patients.

BACKGROUND: The urokinase plasminogen activator (uPA) system is involved in tissue remodelling processes and is up-regulated in many types of malignancies. We investigated whether serum levels of different forms of soluble uPA receptor (suPAR) are associated with survival and in particular with prostate cancer and cardiovascular disease mortality.METHODS: Using time-resolved fluorescence immunoassays, we measured intact suPAR [suPAR(I-III)] and intact plus cleaved suPAR [suPAR(I-III) + suPAR(II-III)] and thus calculated the amount of suPAR(II-III) in serum samples from 375 men participating in a prostate cancer screening trial. A total of 312 men were free of prostate cancer and 63 men had prostate cancer diagnosed at the time of screening. The cohort was followed for 15 years. We assessed survival using Kaplan-Meier estimation and Cox proportional hazards regression.RESULTS: The mean age at blood sampling was 64 years. In total, 152 men died during follow-up. SuPAR(I-III) and suPAR(II-III) were significantly positively associated with mortality (P = 0.001 and P < 0.0001, respectively). In a Cox regression model adjusting for age and prostate cancer status, an increase in suPAR(I-III) and suPAR(II-III) by 1-unit (ln-scale) was associated with a hazard ratio (HR) of 2.26 [95% confidence interval (CI) 1.17-4.35] and 2.53 (95% CI 1.56-4.10), respectively. There was a trend towards an increased risk of death from prostate cancer in screening-detected prostate cancer patients with increased values of either suPAR form. However, this difference was not significant and the association disappeared after adjusting for age, tumour stage, tumour grade and prostate-specific antigen. Being in the highest quartile of any of the suPAR forms was associated with a highly significant increased risk of cardiovascular death, with HR adjusted for age of 3.27 (95% CI 1.38-7.73) for suPAR(I-III) quartile 4 versus quartile 1. Conclusions. High concentrations of serum s

Previous studies have found that soluble urokinase plasminogen activation receptor (suPAR) increases during inflammatory and malignant illness and elevated suPAR levels may be associated with poor clinical outcome. The purpose of this study was to investigate plasma levels of suPAR during the course of allogeneic stem cell transplantation (SCT). Twenty SCT patients were included in the study. suPAR was measured by ELISA in daily taken plasma samples during the pretransplant conditioning with chemotherapy and weekly for 1 month after infusion of the graft. suPAR levels before the start of the conditioning were significantly elevated when compared to those of healthy controls. During the conditioning in particular treatment with antithymocyte globulin was associated with significantly increased suPAR levels (P = 0.012). At day +7 after infusion of the graft, suPAR levels had decreased to pretreatment levels. High suPAR levels at day 0 were associated with increased mortality (P = 0.011). The present study found increased suPAR levels during the conditioning in SCT patients. Further, the data indicated that increased suPAR levels may be associated with increased mortality, suggesting suPAR as a candidate for further studies as an outcome predictor in SCT.

Udgivelsesdato: 2009-Sep-15, BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality in developed countries. It is known that early detection results in improved survival, and consequently there is a need for improved diagnostic tools in CRC. The plasma level of soluble urokinase plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer. METHODS: In a case-control study comprising 308 patients undergoing endoscopical examination following CRC-related symptoms, 77 CRC patients with adenocarcinoma were age and gender matched to: 77 patients with adenomas; 77 with other non-malignant findings, and 77 with no findings. The different uPAR forms were measured in citrate plasma collected before endoscopical examination, using three different Time Resolved - Fluorescence Immuno Assays (TR-FIA's). RESULTS: All soluble uPAR forms were found to be significantly higher in cancer patients than in patients presenting with other non-malignant findings; uPAR(I) P=0.0006, suPAR(I-III) P<0.0001 and suPAR(I-III)+(II-III) P<0.0001, whereas no significant difference was found when performing similar comparisons for patients presenting with adenomas. The odds ratio (OR) for the comparison of uPAR(I) in patients with CRC to subjects with other non-malignant findings was 3.44 (95% CI:1.86-6.37). CRC patients had a mean elevated level of 20.9% (95% CI:10.2-32.6) for suPAR(I-III) and 18.5% (95% CI:9.0-28.8) for suPAR(I-III)+(II-III) compared with subjects with non-malignant findings. CONCLUSIONS: The findings confirm reports on increased uPAR expression in cancer patients and in particular elevated levels of suPAR in blood from CRC patients and indicate that suPAR levels in blood are increasing during carcinogenesis. Although none of the measured uPAR

Udgivelsesdato: 2007-Jan, The blood level of soluble urokinase receptor (suPAR) is increased and associated with a poor clinical or fatal outcome in children with acute malaria. This study hypothesized that the suPAR level would be associated with foetal outcome in maternal malaria. suPAR was measured by ELISA in maternal and cord plasma samples taken during delivery in 253 pregnant Kenyan women stratified according to placental histology: no malaria infection (non-infected), active or active-chronic infection (actively infected) or past-chronic infection (past-infected). Maternal-suPAR was higher in actively infected women (median 3.93 (IQR 2.92-5.29) ng/mL) compared with non-infected (median 2.78 (IQR 1.86-3.87) ng/mL, P = 0.001) and past-infected (median 2.67 (IQR 1.94-3.7) ng/mL, P = 0.012) women. Cord-suPAR was comparable across the groups (median 2.98 (IQR 2.38-3.77) ng/mL). In actively infected women, maternal-suPAR and gestational age were the only independent predictors of birth weight in multivariate linear regression adjusted for maternal-suPAR, HIV-1 infection, age, BMI, haemoglobin, peripheral parasitaemia, parity and gestational age; 1 ng/mL higher maternal-suPAR predicted -56 g (95% CI -100 to -12, P = 0.016) reduced birth weight. Cord-suPAR could not predict birth weight after adjusting for gestational age. Future studies are warranted to investigate whether the maternal suPAR level is increased earlier in pregnancy in women with active placental malaria infection and whether early maternal suPAR measurements can predict birth weight. If so, measurements of maternal suPAR early in pregnancy might then potentially identify women with increased needs for antenatal care and intervention.

Elevated blood levels of soluble urokinase receptor (suPAR) measured by ELISA decrease in human immunodeficiency virus-1 (HIV-1)-infected patients initiating highly active antiretroviral therapy (HAART). As the suPAR ELISA measures both three- and two-domain suPAR [suPAR(I-III), suPAR(II-III)] and suPAR(I-III)-ligand complexes, the amount by which the individual suPAR forms (suPAR(I-III), suPAR(II-III) and one-domain suPAR [suPAR(I)]) decrease in plasma in HIV-1-infected patients initiating HAART is unknown. Consequently, the objective of this study was to investigate HAART-induced changes in the individual plasma suPAR forms in HIV-1-infected patients. Plasma suPAR was measured by three time-resolved fluorescence immunoassays detecting suPAR(I-III), suPAR(I-III) + suPAR(II-III) and suPAR(I) in 29 treatment-naïve HIV-1-infected patients followed annually for 5 years after initiation of HAART and in 20 age- and gender-matched healthy individuals. In addition, plasma levels of the following inflammatory markers were also investigated: soluble tumour necrosis factor receptor (sTNFr)-II, TNF-alpha, interleukins (IL)-10, IL-6, IL-4, IL-2 and interferon (IFN)-gamma. In HIV-1-infected patients, plasma suPAR(I-III), suPAR(II-III) and suPAR(I) decreased within the first treatment year (all P < 0.05) and suPAR(I-III) and suPAR(II-III) remained above normal throughout follow-up (both P < 0.05). Plasma sTNFrII, IL-6, IFN-gamma and IL-10 also decreased during HAART (all P < 0.05). In HIV-1-infected patients, sTNFrII correlated with all suPAR forms before (all P < 0.01) and after 5 years HAART (all P < 0.001), whereas sTNFrII and suPAR did not correlate in healthy individuals. Intact and cleaved plasma suPAR decreased in HIV-1-infected patients initiating HAART but remained above normal. The positive correlation with sTNFrII suggests that the individual plasma suPAR forms are linked to immune activation in HIV-1 infection.

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

Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro in humans. Healthy subjects received intravenous endotoxin injection [high-dose, 2 ng/kg (n=8) and low-dose, 0.06 ng/kg (n=7)], coadministration of 0.06 ng/kg endotoxin and 3 h recombinant human (rh)IL-6 infusion (n=7) or 3 h infusion of rhIL-6 (n=6), rhTNF-alpha (n=6) or NaCl (n=5). Soluble uPAR (suPAR) was measured by enzyme-linked immunosorbent assay in plasma and supernatants from unstimulated and phytohaemagglutinin and lipopolysaccharide-stimulated peripheral blood mononuclear cell (PBMC) cultures incubated for 24 h. The spontaneous and stimulated uPAR-release from PBMC cultures was enhanced 5 h after low-dose endotoxin (both P <0.05), but coadministration of rhIL-6 during low-dose endotoxaemia abolished this enhanced uPAR release. High-dose endotoxin increased plasma suPAR levels (P <0.001) whereas low-dose endotoxin, rhIL-6 or TNF-alpha did not influence uPAR release in vivo to such degree that a systemic effect on the plasma suPAR level was detectable. Even subclinical doses of endotoxin in vivo enhance the capacity of PBMC to release uPAR after incubation in vitro. The inhibitory effect of IL-6 on endotoxin-mediated uPAR-release in vitro suggests that IL-6 has anti-inflammatory effects on endotoxin-mediated inflammation.

The blood levels of the soluble forms of the urokinase receptor (suPAR) are increased in human immunodeficiency virus (HIV)-1-infected patients. This study investigated whether the release of urokinase-type plasminogen activator receptor (uPAR) in whole-blood cultures was affected by HIV infection. The release of different uPAR forms in whole-blood cultures incubated 24 h with or without phytohemagglutinin and lipopolysaccharide was analysed in 47 HIV patients and 19 controls. suPAR was measured by enzyme-linked immunosorbent assay (ELISA) (bulk-suPAR) and three different time-resolved fluorescence immunoassays measuring three-domain suPAR [suPAR(I-III)], three- and two-domain suPAR [suPAR(I-III) + suPAR(II-III)] and one-domain suPAR [suPAR(I)]. The uPAR release was correlated to leucocyte subpopulations and plasma levels of suPAR. The stimulated net whole-blood culture release of bulk-uPAR, uPAR(I-III), uPAR(II-III) and uPAR(I) was reduced in HIV patients (all P < 0.01), whereas the spontaneous bulk-uPAR and uPAR(I-III) release was increased in HIV patients (both P < 0.05). The stimulated uPAR release in whole-blood cultures correlated well to leucocytes and circulating suPAR levels in controls, whereas the correlation was weaker to leucocytes and nonexisting to circulating suPAR levels in HIV patients. These findings demonstrate that HIV infection affects stimulated and spontaneous uPAR release in whole-blood cultures. Given that high blood levels of suPAR in HIV patients are linked to immune activation, the perturbations in uPAR release in whole-blood cultures from HIV patients may also reflect immune activation.

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.