A novel multiplex-protein array for serum diagnostics of colon cancer: a case-control study.

BACKGROUND: More than 1.2 million new cases of colorectal cancer are reported each year worldwide. Despite actual screening programs, about 50% of the patients are diagnosed at advanced tumor stages presenting poor prognosis. Innovative screening tools could aid the detection at early stages and allow curative treatment interventions. METHODS: A nine target multiplex serum protein biochip was generated and evaluated using a training- and validation-set of 317 highly standardized, liquid nitrogen preserved serum samples comprising controls, adenomas, and colon cancers. RESULTS: Serum levels of CEA, IL-8, VEGF, S100A11, MCSF, C3adesArg, CD26, and CRP showed significant differences between cases and controls. The largest areas under the receiver operating characteristics curve were observed for CEA, IL-8, and CRP. At threshold levels yielding 90% specificity, sensitivities for CEA, IL-8 and CRP were 26%, 22%, and 17%, respectively. The most promising marker combinations were CEA + IL-8 reaching 37% sensitivity at 83% specificity and CEA + CRP with 35% sensitivity at 81% specificity. In an independent validation set CEA + IL-8 reached 47% sensitivity at 86% specificity while CEA + CRP obtained 39% sensitivity at 86% specificity. Early carcinomas were detected with 33% sensitivity for CEA + IL-8 and 28% for CEA + CRP. CONCLUSIONS: Apart from CEA, IL-8, and CRP, the screening value of additional blood markers and the potential advantage of combining serum biochip testing with fecal occult blood testing needs to be studied. Multiplex biochip array technology utilizing serum samples offers an innovative approach to colorectal cancer screening.

Predictors of acute kidney injury in patients admitted with ST-elevation myocardial infarction - results from the Bremen STEMI-Registry.

BACKGROUND:: Deterioration of renal function after exposition to contrast media is a common problem in patients with myocardial infarction undergoing percutaneous coronary interventions. The aim of the present study was to assess the incidence of acute kidney injury in patients admitted with ST-elevation-myocardial infarction (STEMI) and its association with infarction severity, comorbidities and treatment modalities, including amount of contrast media applied. METHODS:: All patients with STEMI from the metropolitan area of Bremen, Germany are treated at the Bremen Heart Centre and since 2006 documented in the Bremen STEMI-Registry. Acute kidney injury was graded from stage 0 to 3 following the Kidney-disease-improving-global outcomes criteria from 2012. RESULTS:: Data from 3810 patients admitted with STEMI were included in this study. No acute kidney injury was observed in 3120 (82%) patients while acute kidney injury was detected in 690 (18%) patients: Stage 1: n=497 (13%), 2: n=66 (2%), 3: n=127 (3%). Acute kidney injury was associated with elevated 30-day (0: 3%, 1: 20%, 2: 46%, 3: 58%) and one-year mortality rates (0: 6%, 1: 26%, 2: 49%, 3: 66%). Higher acute kidney injury stages were associated with higher peak creatine kinase (in U/l±SEM): stage 0: 1748±33, 1: 2588±127, 2: 3684±395, 3: 3330±399, p (<0.01), lower mean systolic blood pressure at admission (in mmHG±SD): 0: 133±28, 1: 129±31; 2: 121±31, 3: 115±33 ( p<0.01) and higher Thrombolysis in Myocardial Infarction risk score for STEMI (scale 0-14±SD): 0: 2.71±2, 1: 4.08±2, 2: 4.98±2, 3: 5.05±2, ( p<0.01). However, no such association could be found between acute kidney injury stage and amount of contrast media applied (in ml±SD) 0: 138±57, 1: 139±61; 2: 140±76; 3: 145±80 ( p=0.5). Reduced initial glomerular filtration rate was associated with higher incidences of acute kidney injury while again no relation to amount of contrast media could be observed in subgroups ranked by initial glomerular filtration rate. A multivariate analysis confirmed these results: while left-heart-failure/cardiogenic shock (odds ratio (OR) 4.2, 95% confidence interval (CI) 3.3-5.5) as well as larger infarctions (peak creatine kinase >3000 U/l (OR 2.2, 95% CI 1.7-2.8)) were independently associated with a greater risk for acute kidney injury, amount of contrast media applied during angiography was not (150-250 ml, OR 0.95, 95% CI 0.8-1.2 ( p=0.7), >250 ml, OR 1.3, 95% CI 0.8-2.0 ( p=0.5)). CONCLUSIONS:: Acute kidney injury, which was associated with elevated short- and long-term mortality rates, could be observed in 18% of patients admitted with STEMI. The present data suggest that severity and haemodynamic impairment due to STEMI rather than contrast-media-induced nephropathy is the key contributor for acute kidney injury in STEMI patients. The deleterious effect of the myocardial infarction itself on renal function can be explained through renal hypoperfusion, neurohormonal activation or other pathomechanisms that might have been underestimated in the past.

Comparison of Outcomes of Patients With ST-Segment Elevation Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention Analyzed by Age Groups (<75, 75 to 85, and >85 Years); (Results from the Bremen STEMI Registry).

As old patients, who were treated by percutaneous coronary interventions (PCI) for ST-segment elevation myocardial infarction (STEMI), are regularly excluded or underrepresented in randomized trials, data on treatment and outcomes of this patient group at high risk have to be collected by registries. The study population of the German Bremen STEMI Registry was divided into the age groups G1: <75 years (n = 4,108, young), G2: 75 to 85 years (n = 1,032, old), and G3: >85 years (n = 216, very old) and was evaluated for clinical management and course. PCI failure (Thrombolysis In Myocardial Infarction flow 0 or 1 after PCI) was observed more often with increasing age. Patients >85 years without successful PCI had a very high inhospital mortality (40.0% without PCI success vs 18.1% with PCI success, p <0.05). Despite a reduced rate of periinterventional treatment with glycoprotein IIb/IIIa inhibitors in elderly patients of G2 and G3, inhospital bleedings (Thrombolysis In Myocardial Infarction/Bleeding Academic Research Consortium ≥2) occurred more frequently in these patients (G1: 5.4% vs G2: 11.0% vs G3: 19.6%, p <0.0001). Mortality rates during inhospital and long-term course increased with increasing age. In a multivariate analysis successful PCI was associated with improved outcomes in all age groups; even in very old patients successful PCI was associated with a significantly lower inhospital mortality rate (odds ratio 0.26, 95% confidence interval 0.08 to 0.81) and a trend toward a lower 1-year mortality. In conclusion, the present "real-world" data demonstrate an elevated rate of PCI failure, bleeding complications, and mortality in elderly patients treated by primary PCI for STEMI. However, a beneficial effect of successful PCI on mortality was observed in all age groups, even in very old patients, indicating the crucial role of revascularization therapy.

A novel multiplex-protein array for serum diagnostics of colorectal cancer: impact of pre-analytical storage conditions.

INTRODUCTION: Biomarker discovery studies seldom report on pre-analytical effects. We used a novel multiplex protein biochip for colorectal cancer screening to investigate effects of different storage temperatures and repeated freeze-thaw cycles. METHODS: This biochip, composed of CEA, IL-8, VEGF, M-CSF, S100A11, C3adesArg, CD26, and CRP, was applied to twenty highly standardized preserved serum samples. RESULTS: Aliquot comparison of long-term storage at -80°C (n=20) versus -170°C (n=20) did not show significant differences for any of the eight markers. In contrast, three freeze-thaw cycles (3 × 20 aliquots) detected changes in the serum level for all markers (p<0.05) but S100A11 and CD26: levels of CEA, IL-8, C3adesArg, and CRP increased, while VEGF and M-CSF levels decreased. However, applying diagnostic thresholds for CEA, IL-8, and CRP revealed that freeze-thaw cycles did not affect diagnostic performance. In contrast, analysis of samples stored at -80°C compared to -170°C failed to detect one out of three detectable malignancies. CONCLUSION: We conclude that three freeze-thaw cycles modulated serum marker levels significantly, but do not compromise biochip diagnostic performance. For our marker panel, serum preservation at -80°C seems comparable to -170°C; however, storage at -80°C could lead to misdiagnosis. Our findings emphasize the need for standardized sample collection, processing, storage, and reporting.

Toward standardized high-throughput serum diagnostics: multiplex-protein array identifies IL-8 and VEGF as serum markers for colon cancer.

Development and progression of colon cancer may be related to cytokines. Cytokines with diagnostic value have been identified individually but have not been implemented into clinical praxis. Using a multiplex protein array, the authors explore a panel of cytokines simultaneously and compared its performance to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Serum concentrations of 12 cytokines were simultaneously determined by multiplex biochip technology in 50 colon cancer patients and 50 healthy controls. Serum levels of interleukin-8 (IL-8) and CEA were significantly higher in cancer patients than in healthy controls. Areas under the receiver operating characteristic curves (AUCs) were largest for IL-8, followed by CEA, vascular endothelial growth factor (VEGF), and CA 19-9. Analyses regarding marker combinations showed an advantage over single marker performance for CEA, VEGF, and CA 19-9 but not for IL-8. Multiplex biochip array technology represents a practical tool in cytokine and cancer research when simultaneous determination of different biomarkers is of interest. The results suggest that the assessment of IL-8, CEA, VEGF, and possibly CA 19-9 serum levels could be useful for colon cancer screening with the potential of also detecting early stage tumors. Further validation studies using these and additional markers on a multiplex array format are encouraged.

Comparison of five commercial extraction kits for subsequent membrane protein profiling.

Membrane proteins account for 70-80% of all pharmaceutical targets emphasizing their clinical relevance. Identification of new, differentially expressed membrane proteins reflecting distinct disease properties is thus of high importance. Unfortunately, isolation and analysis of membrane-bound proteins is hampered by their relative low abundance in total cell lysates, their frequently large size and their hydrophobic properties. We thus aimed to identify protocols that allow for highly efficient isolation and purification of membrane-bound proteins for subsequent protein profiling. We present a comparative study of different membrane protein extraction methods that vary in total protein yield between 0.02 and 4.8 mg using constant cell pellets of the colorectal carcinoma cell line SW620. We also demonstrate by means of polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the majority of commercial membrane extraction kits harbor a substantial cytosolic contamination of their membranous fraction. Based on purity of membranous fraction, protein yield, time and costs, we show superiority of two commercial extraction kits for downstream proteome analyses of membrane proteins.