Time-resolved Fluorescence Immunoassays Research Articles

Predictive value of the domain specific PLA2R antibodies for clinical remission in patients with primary membranous nephropathy: A retrospective study.

M-type phospholipase A2 receptor (PLA2R) is a major auto-antigen of primary membranous nephropathy(PMN). Anti-PLA2R antibody levels are closely associated with disease severity and therapeutic effectiveness. Analysis of PLA2R antigen epitope reactivity may have a greater predictive value for remission compared with total PLA2R-antibody level. This study aims to elucidate the relationship between domain-specific antibody levels and clinical outcomes of PMN. This retrospective analysis included 87 patients with PLA2R-associated PMN. Among them, 40 and 47 were treated with rituximab (RTX) and cyclophosphamide (CTX) regimen, respectively. The quantitative detection of -immunoglobulin G (IgG)/-IgG4 targeting PLA2R and its epitope levels in the serum of patients with PMN were obtained through time-resolved fluorescence immunoassays and served as biomarkers in evaluating the treatment effectiveness. A predictive PMN remission possibility nomogram was developed using multivariate logistic regression analysis. Discrimination in the prediction model was assessed using the area under the receiver operating characteristic curve (AUC-ROC).Bootstrap ROC was used to evaluate the performance of the prediction model. After a 6-month treatment period, the remission rates of proteinuria, including complete remission and partial remission in the RTX and CTX groups, were 70% and 70.21% (P = 0.983), respectively. However, there was a significant difference in immunological remission in the PLA2R-IgG4 between the RTX and CTX groups (21.43% vs. 61.90%, P = 0.019). Furthermore, we found differences in PLA2R-CysR-IgG4(P = 0.030), PLA2R-CTLD1-IgG4(P = 0.005), PLA2R-CTLD678-IgG4(P = 0.003), and epitope spreading (P = 0.023) between responders and non-responders in the CTX group. Multivariate logistic analysis showed that higher levels of urinary protein (odds ratio [OR], 0.49; 95% confidence interval [CI], 0.26-0.95; P = 0.035) and higher levels of PLA2R-CTLD1-IgG4 (OR, 0.79; 95%CI,0.62-0.99; P = 0.041) were independent risk factors for early remission. A multivariate model for estimating the possibility of early remission in patients with PMN is presented as a nomogram. The AUC-ROC of our model was 0.721 (95%CI, 0.601-0.840), in consistency with the results obtained with internal validation, for which the AUC-ROC was 0.711 (95%CI, 0.587-0.824), thus, demonstrating robustness. Cyclophosphamide can induce immunological remission earlier than rituximab at the span of 6 months. The PLA2R-CTLD1-IgG4 has a better predict value than total PLA2R-IgG for remission of proteinuria at the 6th month.

Soluble urokinase-type plasminogen activator receptor, suPAR, as a prognostic biomarker in advanced pancreatic cancer.

e16277 Background: High levels of intact and cleaved forms of the urokinase-type Plasminogen Activator Receptor (uPAR) in tissue and blood are associated with a poor prognosis in several types of cancer, including lung, colon, bladder, and cholangiocarcinoma. The soluble forms of uPAR (suPAR) have not been investigated in patients with pancreatic ductal adenocarcinoma (PDAC). The aim was to determine if circulating levels of intact and cleaved suPAR were associated with overall survival (OS) in patients with advanced PDAC. Methods: Patients with PDAC stage 3 or 4 were consecutively included in the BIOPAC study (ClinicalTrials.gov ID: NCT03311776) in a training cohort (n = 230, age 45-89 years, 56% females) from Herlev Hospital and a validation cohort (n = 115, age 40-82 years, 59% females) from four other Danish hospitals between 2008 to 2014. A total of 95% of the patients in the training cohort and 93% in the validation cohort received first-line palliative chemotherapy. One patient was alive at the time of analysis at 8 years follow-up. Serum samples were collected prior to palliative chemotherapy. Different suPAR forms were determined using time-resolved fluorescence immunoassays (TR-FIA). The Cox proportional hazards model was used for uni- and multivariate analyses. Age, performance status, stage (4 vs 3), gender, Charlson age-adjusted comorbidity index, and CA19-9 were included in the multivariate analysis for OS. Results: In the training cohort, high level of suPAR domain I was an independent biomarker of short OS (HR, 1.37, 95% CI 1.15-1.63, p = 0.0004). High CA19-9 (HR, 1.08, 95% CI 1.04-1.12, p < 0.0001), PS, and stage were also independent prognostic parameters in the training cohort. In the validation cohort, high level of suPAR domain I (HR, 1.91, 95% CI 1.36-2.69, p = 0.0002) and CA 19-9 (HR, 1.09, 95% CI 1.01-1.17, p = 0.02) were confirmed as independent prognostic biomarkers for short OS. No correlation was observed between suPAR domain 1 and CA19-9 (r = 0.059, p = 0.21). Conclusions: A high pretreatment circulating level of suPAR domain I is associated with a short OS in patients with advanced PDAC, and the biomarker is independent of CA19-9. The results warrant further investigation of suPAR domain 1 as a biomarker in patients with PDAC.

Quantitative detection of anti-PLA2R antibodies targeting different epitopes and its clinical application in primary membranous nephropathy.

This study aimed to establish time-resolved fluorescence immunoassays to quantitatively detect the autoantibodies targeting different epitopes of M-type phospholipase A2 receptor (PLA2R) and evaluate its clinical application in primary membranous nephropathy (PMN). PLA2R and its reactive epitope-specific IgG/IgG4 time-resolved fluorescence immunoassays (TRFIAs) were established using europium-labeled anti-human IgG/IgG4 antibodies, recombinant proteins, and patient serum. The levels of IgG/IgG4 targeting PLA2R and its epitopes in PMN patient serum were detected, and the relationship between epitope spreading of PLA2R and the severity of patients with PMN was evaluated. The TRFIAs established in this study could quantitatively detect PLA2R and its epitope-specific IgG and IgG4. Sera from 59 patients with PMN were subjected todetection using anti-PLA2R IgG and anti-PLA2R IgG4. Among them, 46 and 54 patients were found positive forPLA2R antibodies, respectively. Moreover, the levels of PLA2R antibodies were strongly correlated with the severity of patients with PMN. Patients who were detected to have two or more epitopes had more serious renal injury. PLA2R domain-specific IgG/IgG4 TRFIAs were established in this study, and detection with anti-PLA2R IgG4 could more sensitively screen the reactivity of patients to the PLA2R domain. Moreover, detection epitope spreading of PLA2R was confirmed which is related to the severity of patients with PMN.

Testis development in the Japanese eel is affected by photic signals through melatonin secretion.

ObjectiveAccording to reported spawning characteristics of Japanese eel, Anguilla japonica, which exhibit spawning and migration patterns that are synchronized with lunar cycles and photoperiod, we hypothesized that a close association exists between specific photic signals (daylight, daylength, and moonlight) and endocrinological regulation. Given the photic control in melatonin secretion, this hypothesis was tested by investigating whether melatonin signals act as mediators relaying photic signals during testis development in the eel.MethodsWe examined changes in melatonin-secretion patterns using time-resolved fluorescence immunoassays in sexually immature and mature male Japanese eels under the condition of a new moon (NM) and a full moon (FM).ResultsThe eye and plasma melatonin levels exhibited a nocturnal pattern under a 12-h light: dark cycle (12L12D) or under constant darkness (DD), but not with constant light (LL). Eye melatonin levels were similar under the 12L12D and short-day (9L15D) conditions. In the long-day condition (15L9D), secreted plasma melatonin levels were stable, whereas short-day melatonin secretion began when darkness commenced. Sexual maturation began at 8 weeks following intraperitoneal injection of human chorionic gonadotropin (hCG), and NM exposure led to significantly higher eye and plasma melatonin levels compared with those detected under FM exposure.

Circulating Forms of Urokinase-Type Plasminogen Activator Receptor in Plasma Can Predict Recurrence and Survival in Patients with Urothelial Carcinoma of the Bladder.

Simple SummaryBladder cancer is an aggressive disease and after operation many patients are at risk of recurrence and shortened survival. Specific proteins are known to be of importance in the development of cancers. One of these proteins is the urokinase-type plasminogen activator receptor (uPAR) which exists in different forms. We here investigate the presence of the different uPAR forms in plasma from patients with bladder cancer, and we associate the elevated amount of uPAR forms with survival. We find that high levels of all uPAR forms is associated with short survival in patients with bladder cancer and suggest that this in the future might help improve handling of the disease.Urothelial carcinoma of the bladder is a highly aggressive disease characterised by a very heterogeneous clinical outcome. Despite cystectomy, patients still have a high recurrence risk and shortened survival. Urokinase-type plasminogen activator receptor (uPAR) is present in tumour tissue specimens from patients with urothelial carcinoma. The different uPAR forms in blood are strong prognostic markers in other cancer types. We investigate the presence of different uPAR forms in tumour tissue and test the hypothesis that preoperative plasma levels of the uPAR forms predict recurrence free survival, cancer specific survival, and overall survival in patients treated with cystectomy for urothelial carcinoma. Using Western blotting we analyse neoplasia and adjacent benign-appearing urothelium from randomly selected patients for the presence of intact and cleaved uPAR forms. Prospectively collected preoperative plasma samples from 107 patients who underwent radical cystectomy for urothelial carcinoma are analysed. The different uPAR forms are measured by time-resolved fluorescence immunoassays. uPAR in tumour tissue from patients with urothelial carcinoma is demonstrated in both an intact and cleaved form. The different uPAR forms in plasma are all significantly associated with both recurrence free survival, cancer specific survival, and overall survival, high concentrations predicting short survival. uPAR (I) has the strongest association with a HR of 2.56 for overall survival. In the multivariable survival analysis uPAR (I) is significantly associated with cancer specific survival and overall survival.

Sensitive Time-Resolved Fluorescence Immunoassay for Quantitative Determination of Oxyfluorfen in Food and Environmental Samples.

The direct and indirect competition time-resolved fluorescence immunoassays (dc-TRFIA, ic-TRFIA) were established by combining the autofluorescence properties of lanthanide europium (Eu) with the monoclonal antibody of oxyfluorfen. The purified Eu antibody was optimized and the conditions such as the working concentration of the Eu antibody, monoclonal antibody, and working buffer were optimized. In the optimal condition, the IC50 of dc-TRFIA was 10.27 ng/mL, the lowest detection limit IC10 was 0.071 ng/mL, the detection range (IC10-IC90) was 0.071–1074.3 ng/mL, and the detection range (IC10-IC90) and IC50 of ic-TRFIA were 0.024–504.6 and 2.76 ng/mL, respectively. The comparison showed that the sensitivity and detection limit of ic-TRFIA were superior to dc-TRFIA. The cross reaction (CR) tests showed that the CR with other oxyfluorfen structure analogs was <0.02%, except that there was a certain CR with the benzofluorfen (CR = 11.58) and the bifenox (CR = 8.23%). The average recoveries of ic-TRFIA were 74.6–108.3%, and the RSDs were between 2.1 and 10.9%, in the addition recovery test with five substrates. The results of the correlation test with the real samples of GC-ECD showed that they were highly correlated (y = 0.975x – 0.4446, R2 = 0.9901), which proved that the TRFIA method established in this study had high reliability and accuracy and could be used in environment and agricultural products for rapid detection of oxyfluorfen residues.

Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR.

Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules for therapeutics and biotechnological applications. Most widely employed chemical labeling methods for proteins are based on targeting of Lys or Cys residues that leads to mixed stoichiometry in the degree of conjugation and may interfere with antigen binding, thus, compromising the function of the antibody. A site-specific oligonucleotide conjugation technology providing full control over valency in mild reaction conditions would be an advancement to the state-of-the-art in bioconjugation. Herein, we demonstrate the production of single-chain variable fragment antibodies with fused SpyCatcher (scFv-SpyCatcher, monovalent) and alkaline phosphatase-SpyCatcher (scFv-AP-SpyCatcher, bivalent) on C-terminus and their conjugation to SpyTag002-oligonucleotide in phosphate-buffered saline (PBS). The formation of a covalent isopeptide bond between the protein and SpyTag002-oligonucleotide was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the functionality of the obtained AOCs was confirmed in immuno-polymerase chain reaction (PCR) assays for the detection of microcystin-LR and 17β-estradiol. Based on time-resolved fluorescence immunoassays with scFv-AP fusion constructs, we observed that the SpyCatcher and SpyCatcher-SpyTag002-oligonucleotide part lowered the absolute signal obtained from the assay by 27.6 and 48.4% at 2 nM and by 26.2 and 27.6% at 100 pM microcystin-LR and 17β-estradiol concentrations, respectively. Nevertheless, the overall sensitivity of the immuno-PCR assays was similar to the time-resolved fluorescence immunoassays performed with the same components. In this study, vectors for SpyCatcher-fusion construction were created for directional cloning with SfiI sites enabling the rapid generation of AOC constructs for site-specific SpyTag-oligonucleotide conjugation.

Simultaneous competitive and sandwich formats multiplexed immunoassays based on ICP-MS detection

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) based immunoassay method has been proposed in multiple immunoassays but has not been used in competitive and sandwich formats immunoassay simultaneously. The two immunoassays were usually conducted separately in clinical field depending on the size and the amount of binding sites of targets. We proposed an immunoassay method based on magnetic beads and ICP-MS detection that could be suitable for both small and large molecules. The functionalized magnetic beads were added to capture the immune complex after immune reaction. In this way, thyrotropin and free thyroid hormone can be captured, separated and then detected according to the elemental tags by ICP-MS simultaneously. The new method was evaluated by comparing the results with time resolved fluorescence immunoassays (TRFIA). The dynamic ranges of TSH and FT4 were 0.16–105 mU/L and 3.5–65 pmol/L, respectively. The limits of detections were 0.06 mU/L for TSH and 1.59 pmol/L for FT4. And the relative standard deviations (RSD) of TSH and FT4 were 4.64% at 2.5 mU/L and 1.87% at 5.85 pmol/L. This immunoassay method enables the determination of small and large biomolecules simultaneously via competitive and sandwich immunoassay formats.

Plasma levels of intact and cleaved urokinase plasminogen activator receptor (uPAR) in men with clinically localised prostate cancer

AimsLymph node metastasis (N1) is an adverse prognostic factor for men with clinically localised prostate cancer (PCa), but the prediction of N1 disease remains difficult. Urokinase plasminogen activator receptor (uPAR)...

Urokinase plasminogen activator receptor (uPAR) as a novel biomarker in prostate cancer.

183 Background: New biomarkers are warranted to distinguish between indolent and aggressive prostate cancer (PCa). The urokinase plasminogen activator receptor (uPAR) plays an important role in pericellular proteolysis by binding urokinase plasminogen activator (uPA). This is required for degradation of the extracellular matrix and for cancer invasion. In addition to binding uPAR, uPA cleaves uPAR liberating uPAR(I) and uPAR(II-III). Intact, uPAR(I-III), and uPAR(II-III) can be liberated from the cell surface resulting in three different uPAR forms in circulation. The different uPAR forms are strong prognostic markers in several cancers. We measured the uPAR forms in plasma from patients with different stages of PCa. Methods: Between February 1, 2012 and October 1, 2014, 400 patients with PCa (se table) had plasma samples obtained. The levels of intact uPAR [uPAR(I-III)], intact uPAR + cleaved uPAR domains II+III [uPAR(I-III) + uPAR(II-III)], and cleaved uPAR domain I [uPAR(I)] were determined in citrated plasma samples with two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs). Results: Plasma uPAR(I-III) + uPAR(II-III) and uPAR(I) were significantly higher in hormone naïve patients and CRPC patients compared to patients with localized disease (see table). Quantification of intact uPAR(I-III) revealed no significant differences between the three groups. Conclusions: Our findings suggest that uPAR(I-III) + uPAR(II-III) and uPAR(I) are associated with higher tumor stage as well as advanced PCa disease. These associations suggest that uPAR domains in plasma harbour prognostic value for PCa patients. Further studies are warranted to validate the use of uPAR forms as biomarkers for PCa. [Table: see text]

The soluble urokinase plasminogen activator receptor and its fragments in venous ulcers.

Activation of proteolytic mechanisms at the cell surface through the activity of urokinase-type plasminogen activator (uPA) bound to its receptor, uPAR, is an important process in wound healing. The soluble forms of uPAR (suPAR and its fragments I, II, and III) have nonproteolytic functions that include chemotaxis, adhesion, and proliferation, which also have a role in wound healing. The aim of this study was to determine whether suPAR and its cleaved fragments are present in venous ulcers and whether their levels are associated with healing. Ulcer exudates were collected from patients with venous leg ulcers (n = 30). Healing was defined as complete re-epithelialization within 6 months of compression therapy. Time-resolved fluorescence immunoassays were validated for quantification of suPAR and its fragments in ulcer exudates. The effect of exudates on keratinocyte migration was analyzed by an in vitro scratch assay. Ulcer exudates from patients who healed (n = 9) had approximately threefold higher levels of intact suPAR (P = .005), twofold higher levels of suPARI (P = .03), and approximately threefold higher levels suPARII-III (P < .0001) compared with nonhealers (n = 21). Exudate from healing ulcers stimulated keratinocyte migration (P = .02), whereas depletion of suPAR from exudates resulted in cell apoptosis. We conclude that suPAR and its fragments are present in the environs of venous ulcers and may act as indicators of the propensity of venous ulcers to heal, with suPARII-III being the best discriminator. We speculate that suPAR and its fragments may have a role in the maintenance of an optimal ulcer-healing environment.

Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor: Biological variation, reference intervals and clinical useful cut-points

BackgroundHigh levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis. MethodsuPAR forms were measured in citrated and EDTA plasma samples using time-resolved fluorescence immunoassays. Diurnal, intra- and inter-individual variations were assessed in plasma samples from cohorts of healthy individuals. Reference intervals were determined in plasma from healthy individuals randomly selected from a Danish multi-center cross-sectional study. A cohort of CRC patients was selected from the same cross-sectional study. ResultsThe reference intervals showed a slight increase with age and women had ~20% higher levels. The intra- and inter-individual variations were ~10% and ~20–30%, respectively and the measured levels of the uPAR forms were within the determined 95% reference intervals. No diurnal variation was found. Applying the normal upper limit of the reference intervals as cut-point for dichotomizing CRC patients revealed significantly decreased overall survival of patients with levels above this cut-point of any uPAR form. ConclusionsThe reference intervals for the different uPAR forms are valid and the upper normal limits are clinically relevant cut-points for CRC prognosis.

Prognostic significance of circulating intact and cleaved forms of urokinase plasminogen activator receptor in inoperable chemotherapy treated cholangiocarcinoma patients

BackgroundHigh 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 methodsPatients 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. ResultsBaseline 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). ConclusionThe baseline level of uPAR(I–III)+uPAR(II–III) is a predictor of survival in inoperable cholangiocarcinoma patients.

Rapid Homogenous Time-Resolved Fluorescence (HTRF) Immunoassay for Anthrax Detection

Infection with Bacillus anthracsis spores induces an acute anthrax disease that can cause casualties and death in untreated cases. Thus rapid diagnosis of anthrax at early stage of the disease is essential to allow an effective treatment. Here we present the development of rapid and sensitive homogenous time-resolved fluorescence (HTRF) immunoassays based on the energy transfer process of europium cryptate (EuK) donor to AlexaFluor647 acceptor. The energy transfer process is limited to d < 10nm, making the HTRF an ideal assay for examination of homogenous and complex samples, since only mutual binding of the donor and acceptor antibodies to the analyte would result in positive signal. HTRF assay was developed for the detection of the bacterial Protective Antigen (PA) toxin, a serological marker that correlates with bacteremia in infected hosts, using two monoclonal anti-PA antibodies that specifically recognize two different epitopes on the PA molecule. The assay was sensitive enabling detection of 2ng/ml PA in the serum of B. anthracsis-infected rabbits in only 15min assay. Additionally, HTRF assay was developed for the detection of bacterial spores using polyclonal anti-spore antibodies that recognize many epitopes on the bacterial surface. The assay enabled the detection of 2 × 10(6) spores/ml in 30min assay and was specific, showing no cross reactivity with closely related non-virulent bacillus cereus strain. This study describes the use of the HTRF assay for the detection of both singled-epitope (proteins) and multi-epitope (particles) as rapid, simple and sensitive method that can be used at the time that fast results are needed to allow an effective medical care.

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.

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

ObjectivesTo 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 methodsSerum 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). ResultsHigh 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. ConclusionsThis 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.

Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening

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. 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. 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 suPAR(I-III) and suPAR(II-III) were associated with poor overall survival. The association was particularly strong for death from cardiovascular disease. No similar association was found for prostate cancer after adjustment for other prognostic factors.

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 uPAR forms were measured in samples from 131 metastatic PC patients. These constituted a subset of patients included in a randomized clinical trial of treatment with total androgen blockade (TAB) versus polyestradiol phosphate (PEP). Pre-treatment serum levels of intact uPAR (uPAR(I-III)), intact plus cleaved uPAR (uPAR(I-III) + uPAR(II-III)) and domain I (uPAR(I)) were measured using time-resolved fluorescence immunoassays (TR-FIAs). High serum levels of each of the uPAR forms were significantly associated with short overall survival (OS). The prognostic impact was strongest in the TAB treated patients with all uPAR forms being statistically significant. In multivariate analysis, uPAR(I-III) + uPAR(II-III) was an independent prognostic factor in TAB treated patients (HR = 5.2, 95% confidence interval (CI): 2.5-10.6, P < 0.0001) but not in PEP treated patients (P = 0.40). In the entire study population, OS was similar in the two treatment groups. The survival analysis showed significant interactions between treatment modality and the level of either uPAR(I-III) or uPAR(I-III) + uPAR(II-III). High levels of uPAR(I-III) + uPAR(II-III) were found to be predictive of effect of PEP versus TAB treatment. Patients with uPAR(I-III) + uPAR(II-III) levels above the median had significantly longer OS (median difference 11.3 months), if treated with PEP rather than with TAB (HR = 1.8, 95% CI:1.1-3.1, P = 0.03). uPAR forms are significantly associated with OS. High uPAR(I-III) + uPAR(II-III) predicts longer OS in patients treated with PEP compared to TAB. uPAR forms are promising prognostic and predictive markers in PC.

Prognostic value of intact and cleaved forms of the urokinase plasminogen activator receptor in a retrospective study of 518 colorectal cancer patients

Background. The levels of the soluble urokinase plasminogen activator receptor (suPAR) in blood have been shown to correlate with prognosis in various cancers. Plasma levels of the combined suPAR forms have previously shown to be a strong prognostic marker in the present cohort of CRC patients and could potentially identify high-risk patients among those with early stage disease. In order to investigate whether the individual suPAR forms are stronger prognostic markers than the combined amount we measured the different uPAR forms in serum from the same cohort and evaluated their prognostic significance. Material and methods. The different suPAR forms were measured in serum preoperatively collected from 518 patients. Patients were followed up to nine years (median 7.9 years) and the primary endpoint was overall survival. The different suPAR forms were measured using Time Resolved Fluorescence Immunoassays(TR-FIAs): Intact, suPAR(I–III) by TR-FIA 1; intact and cleaved, suPAR(I–III)+(II–III) by TR-FIA 2; and liberated uPAR(I) by TR-FIA 3. Results. All three uPAR variants demonstrated prognostic significance when evaluated individually. In a multivariable analysis suPAR(I–III)+(II–III) and the liberated uPAR(I) were shown to be independent markers of prognosis (HR=1.74, CI:1.33–2.26; p <0.0001 and HR=1.32; CI:1.02–1.71; p=0.036 respectively), and independent of the clinical baseline variables: age, gender, tumor stage and localization. Conclusion. This study demonstrated that suPAR(I–III)+(II–III) and the liberated uPAR(I) in serum are independent prognostic markers in CRC.

Growth hormone isoforms in acromegalic patients before and after treatment with octreotide LAR

Background Human growth hormone (hGH) circulates as a mixture of different isoforms. It has been previously reported that the ratio of 20 kDa to 20 kDa plus 22 kDa (%20 kDa-hGH) is increased in patients with active acromegaly. Objectives To evaluate the GH isoforms (20 kDa- and 22 kDa-hGH) in acromegalic patients before and after six months of treatment with octreotide LAR, and to compare the results with those in healthy controls. In addition, the relationships between the %20 kDa-hGH, tumor size and biochemical measurements were also investigated. Design Random serum samples from 23 acromegalic patients evaluated before and after six months of treatment with octreotide LAR and from 23 matched healthy controls were studied. Growth hormone, IGF-I and prolactin (PRL) were measured by chemiluminescence immunometric assay and the 20 kDa- and 22 kDa-hGH isoforms were measured by specific time-resolved fluorescence immunoassays. Results In acromegalic patients before treatment, there was a significantly higher median %20 Da-hGH in comparison to healthy controls (14.31% vs. 9.59%, p < 0.001). After six months of treatment, the median %20 kDa-hGH was similar to the baseline values. Patients with GH < 2.5 ng/mL after six months of treatment had already lower GH and %20 kDa-hGH at baseline ( p < 0.01). The IGF-I (SD-scores) was positively correlated to total GH levels in acromegalic patients after treatment. There was no correlation between the %20 kDa-hGH and PRL levels or tumor size. Conclusions Our study confirmed that acromegalic patients have an increased proportion of circulating 20 kDa-hGH isoform. Consequently, the use of a 22 kDa-hGH specific assay may underestimate the tumor production of total GH. Although octreotide LAR promoted a significant decrease in the GH and IGF-I levels, it did not normalize the GH isoforms composition and suggests that the secretion of GH isoforms is equally inhibited by somatostatin analogues and that it is the disease control that normalizes the GH isoforms composition in acromegaly.