Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins

Glucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression

New strategy for the identification of prostate cancer: The combination of Proclarix and the prostate health index

Prostate health index (PHI) and, more recently, Proclarix have been proposed as serum biomarkers for prostate cancer (PCa). In this study, we aimed to evaluate Proclarix and PHI for predicting clinically significant prostate cancer (csPCa)

Evaluation of Proclarix in the diagnostic work‐up of prostate cancer

Objectives: The use of multiparametric magnetic resonance imaging (mpMRI) has been widely adopted in the diagnostic work‐up for suspicious prostate cancer (PCa) and is recommended in most current guidelines. However, mpMRI lesions are often indeterminate and/or turn out to be false‐positive on prostate biopsy. The aim of this work was to evaluate Proclarix, a biomarker test for the detection of relevant PCa, regarding its diagnostic value in all men before biopsy and in men with indeterminate lesions on mpMRI (PI‐RADS 3) during work‐up for PCa. Materials and Methods: Men undergoing mpMRI‐targeted and systematic biopsy of the prostate were prospectively enrolled. The Proclarix test was evaluated for the detection accuracy of clinically significant PCa (csPCa) defined as Grade Group ≥ 2 and its association to mpMRI results. Further, Proclarix's performance was also tested when adapted to prostate volume (Proclarix density) and performance compared to PSA density (PSAD). Results: A total of 150 men with a median age of 65 years and median PSA of 5.8 ng/mL were included in this study. CsPCa was diagnosed in 65 (43%) men. Proclarix was significantly associated with csPCa and higher PI‐RADS score (p < 0.001). At the pre‐defined cut‐off of 10%, Proclarix's sensitivity for csPCa was 94%, specificity 19%, negative predictive value 80% and positive predictive value 47%. Proclarix density showed the highest AUC for the detection of csPCa of 0.77 (95%CI: 0.69–0.85) compared to PSA, PSAD and Proclarix alone. Proclarix was able to identify all six csPCa in men with PI‐RADS 3 lesions (n = 28), whereas PSAD missed two out of six. At optimized cut‐offs, Proclarix density outperformed PSAD by potentially avoiding 41% of unnecessary biopsies. Conclusion: Proclarix demonstrates high sensitivity in detecting csPCa but may still result in unnecessary biopsies. However, Proclarix density was able to outperform PSAD and Proclarix and was found to be useful in men with PI‐RADS 3 findings by safely avoiding unnecessary biopsies without missing csPCa

Similarities and Differences of Blood N-Glycoproteins in Five Solid Carcinomas at Localized Clinical Stage Analyzed by SWATH-MS.

Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitate follow-up biomarker research into early cancer detection

On the development of plasma protein biomarkers

The development of plasma biomarkers has proven to be more challenging than initially anticipated. Many studies have reported lists of candidate proteins rather than validated candidate markers with an assigned performance to a specific clinical objective. Biomarker research necessitates a clear rational framework with requirements on a multitude of levels. On the technological front, the platform needs to be effective to detect low abundant plasma proteins and be able to measure them in a high throughput manner over a large amount of samples reproducibly. At a conceptual level, the choice of the technological platform and available samples should be part of an overall clinical study design that depends on a joint effort between basic and clinical research. Solutions to these needs are likely to facilitate more feasible studies. Targeted proteomic workflows based on SRM mass spectrometry show the potential of fast verification of biomarker candidates in plasma and thereby closing the gap between discovery and validation in the biomarker development pipeline. Biological samples need to be carefully chosen based on well-established guidelines either for candidate discovery in the form of disease models with optimal fidelity to human disease or for candidate evaluation as well-designed and annotated clinical cohort groups. Most importantly, they should be representative of the target population and directly address the investigated clinical question. A conceptual structure of a biomarker study can be provided in the form of several sequential phases, each having clear objectives and predefined goals. Furthermore, guidelines for reporting the outcome of biomarker studies are critical to adequately assess the quality of the research, interpretation and generalization of the results. By being attentive to and applying these considerations, biomarker research should become more efficient and lead to directly translatable biomarker candidates into clinical evaluation

Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.

Although the classification of cell types often relies on the identification of cell surface proteins as differentiation markers, flow cytometry requires suitable antibodies and currently permits detection of only up to a dozen differentiation markers in a single measurement. We use multiplexed mass-spectrometric identification of several hundred N-linked glycosylation sites specifically from cell surface-exposed glycoproteins to phenotype cells without antibodies in an unbiased fashion and without a priori knowledge. We apply our cell surface-capturing (CSC) technology, which covalently labels extracellular glycan moieties on live cells, to the detection and relative quantitative comparison of the cell surface N-glycoproteomes of T and B cells, as well as to monitor changes in the abundance of cell surface N-glycoprotein markers during T-cell activation and the controlled differentiation of embryonic stem cells into the neural lineage. A snapshot view of the cell surface N-glycoproteins will enable detection of panels of N-glycoproteins as potential differentiation markers that are currently not accessible by other means

Analytical performance of thrombospondin-1 and cathepsin D immunoassays part of a novel CE-IVD marked test as an aid in the diagnosis of prostate cancer.

The Prostate Specific Antigen (PSA) test suffers from low specificity for the diagnosis of Prostate Cancer (PCa). We originally discovered two cancer-related proteins thrombospondin-1 (THBS1) and cathepsin D (CTSD) using a mass-spectrometry-based proteomics approach. The two serum proteins were shown to improve the diagnosis of high-grade PCa. Thus, we developed quantitative ELISAs for the determination of their concentration in human serum. Here we report their analytical performance in terms of limit of detection, specificity, precision, linearity and interferences, which were determined based on CLSI guidelines. Further, we investigated the influence of pre-analytical factors on concentration measurements. For this, blood from 4-6 donors was collected in different tubes and stored at room temperature for different times prior to centrifugation at different centrifugal forces and temperatures. Stability of THBS1 and CTSD under different storage temperatures was also evaluated. Our results show that the assays are specific, linear and sensitive enough to allow measurement of clinical samples. Precision in terms of repeatability and total within-laboratory coefficient of variation (CV) are 5.5% and 8.1% for THBS1 and 4.3% and 7.2% for CTSD, respectively. Relative laboratory-to-laboratory differences were -6.3% for THBS1 and -3% for CTSD. Both THBS1 and CTSD were stable in serum samples, with 80-120% recoveries of concentrations across donors, sample preparation and storage. In conclusion, the ELISAs as part of the novel commercial in vitro diagnostic test Proclarix are suitable for the use in clinical practice. THBS1 and CTSD can be accurately measured for their intended use independent of the lot and laboratory when conditions consistent with routine practice for PSA sampling and storage are used

Use of MS-GUIDE for identification of protein biomarkers for risk stratification of patients with prostate cancer

BACKGROUND Non-invasive liquid biopsies could complement current pathological nomograms for risk stratification of prostate cancer patients. Development and testing of potential liquid biopsy markers is time, resource, and cost-intensive. For most protein targets, no antibodies or ELISAs for efficient clinical cohort pre-evaluation are currently available. We reasoned that mass spectrometry-based prescreening would enable the cost-effective and rational preselection of candidates for subsequent clinical-grade ELISA development. METHODS Using Mass Spectrometry-GUided Immunoassay DEvelopment (MS-GUIDE), we screened 48 literature-derived biomarker candidates for their potential utility in risk stratification scoring of prostate cancer patients. Parallel reaction monitoring was used to evaluate these 48 potential protein markers in a highly multiplexed fashion in a medium-sized patient cohort of 78 patients with ground-truth prostatectomy and clinical follow-up information. Clinical-grade ELISAs were then developed for two of these candidate proteins and used for significance testing in a larger, independent patient cohort of 263 patients. RESULTS Machine learning-based analysis of the parallel reaction monitoring data of the liquid biopsies prequalified fibronectin and vitronectin as candidate biomarkers. We evaluated their predictive value for prostate cancer biochemical recurrence scoring in an independent validation cohort of 263 prostate cancer patients using clinical-grade ELISAs. The results of our prostate cancer risk stratification test were statistically significantly 10% better than results of the current gold standards PSA alone, PSA plus prostatectomy biopsy Gleason score, or the National Comprehensive Cancer Network score in prediction of recurrence. CONCLUSION Using MS-GUIDE we identified fibronectin and vitronectin as candidate biomarkers for prostate cancer risk stratification