Stefan Palme

Identification of Nicotinamide N-Methyltransferase as a Novel Serum Tumor Marker for Colorectal Cancer

Purpose: The goal of this study was to identify and validate novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm. Experimental Design: Employing two-dimensional gel electrophoresis and mass spectrometry, we analyzed 16 matched colorectal cancer and adjacent normal tissue samples. Proteins found to be elevated in cancer tissue were further validated by generating antibodies which were used for immunoblotting of tissue samples and for the development of highly sensitive immunoassays for assessment of serum samples. Results: In total, 735 different proteins were identified in colon tissue. Strong elevation in colorectal cancer for five proteins was confirmed by immunoblot analysis: transforming growth factor-β induced protein ig-h3 (βIG-H3), nicotinamide N-methyltransferase (NNMT), nucleoside diphosphate kinase A (nm23-H1), purine nucleoside phosphorylase (PNPH), and mannose-6-phosphate receptor binding protein 1 (M6P1). Elevated levels of NNMT, which is not predicted to be secreted but is known as a cytoplasmic protein, were found in serum from patients with colorectal cancer. Employing a receiver-operating characteristic curve based on the measurement of 109 patients with colorectal cancer and 317 healthy controls, we obtained an area under the curve of 0.84 for NNMT, which was superior to the established tumor marker carcinoembryogenic antigen with an area under the curve of 0.78. Conclusions: It is proposed that NNMT serum levels may have significance in the early detection and in the management of patients with colorectal cancer.

Identification of PSME3 as a Novel Serum Tumor Marker for Colorectal Cancer by Combining Two-dimensional Polyacrylamide Gel Electrophoresis with a Strictly Mass Spectrometry-based Approach for Data Analysis

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550–6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.

The Extracellular Human Melanoma Inhibitory Activity (Mia) Protein Adopts an SH3 Domain-Like Fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain‐like fold in solution, with two perpendicular, antiparallel, three‐ and five‐stranded β‐sheets. In contrast to known structures with the SH3 domain fold, MIA is a single‐domain protein, and contains an additional antiparallel β‐sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain‐like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin α4β1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Deconstruction of a Nutlin: Dissecting the Binding Determinants of a Potent Protein–Protein Interaction Inhibitor

Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments.

Towards a comprehensive proteome of normal and malignant human colon tissue by 2‐D‐LC‐ESI‐MS and 2‐DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.

Multicenter evaluation of analytical characteristics of the Elecsys® Periostin immunoassay

OBJECTIVE The multifunctional cytokine IL-13 is thought to play a central role in Type 2 inflammation in asthma. Serum periostin has been explored as a candidate biomarker for evaluating IL-13 activity in the airway. We describe the technical performance characteristics of a novel, fully automated immunoassay for the determination of periostin in serum. DESIGN AND METHODS Limit of blank [LoB], limit of detection [LoD] and limit of quantitation [LoQ], linearity, precision and reproducibility across sites and lots were evaluated according to Clinical and Laboratory Standards Institute guidelines. Interferences and sample stability were also investigated. RESULTS The pre-specified values for LoB (2ng/mL), LoD (4ng/mL) and LoQ (10ng/mL) were met. The assay was linear throughout the measuring range (10-160ng/mL) with recoveries within ±10% of target at concentrations >30ng/mL and within ±3ng/mL at concentrations ≤30ng/mL. Recovered periostin concentrations were also within ±10% of target in presence of 43 potentially interfering substances and drugs. Samples were stable across various storage conditions and durations (24h at room temperature, 7days at 4°C, 12weeks at -20°C, and 3 freeze/thaw cycles). Repeatability experiments resulted in CVs across samples and controls ranging from 0.9-1.5%. Intermediate precision was 1.2-1.7% and reproducibility including 3 testing sites and 3 reagent lots was 1.7-3.1%. The final assay correlates to the assay version used in previous clinical trials (Pearsons r=0.998, bias at 50ng/mL=1.2%). CONCLUSION The performance evaluation of the Elecsys® Periostin immunoassay including a multicenter precision analysis demonstrated that the assay is suitable for measuring serum periostin at clinically important concentrations around 50ng/mL.