Roland Kellner

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter‐ and intra‐assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Design of proteome-based studies in combination with serology for the identification of biomarkers and novel targets.

Recently proteome analysis has rapidly developed in the post‐genome era and is now widely accepted as a complementary technology to genetic profiling. The improvement in the technology of both two‐dimensional electrophoresis (2‐DE) analysis as well as protein identification has made proteomics a valuable and powerful tool to study human diseases. A combination of conventional proteome analysis with serology has been developed as a promising experimental approach for the discovery of serological markers in different malignancies. However, the design of proteome‐based studies has to be carefully performed since there are a number of critical needs for systematic and reproducible proteome analysis. In particular, the selection of tissue and its preparation represent an important step in proteome analysis. Besides the preparation of protein samples, the 2‐DE and protein identification is a further critical issue. So far proteome‐based technologies have been successfully used in tumor immunnology for the identification of tumor‐specific autoantigens. Similarly, this technology has been employed for the detection of virulence factors, antigens and vaccine candidates in infectious diseases, as well as for the identification of diagnostic and prognostic markers, suggesting that proteome‐based analysis is a promising tool for the identification of prognostic, diagnostic markers as well as for novel therapeutic targets which could be used for treatment of diseases. The integration of proteome‐based approaches with data from genomic or genetic profiling will lead to a better understanding of different diseases, which will then contribute to the direct translation of the research findings into clinical practice.

Targeting of tumor associated antigens in renal cell carcinoma using proteome‐based analysis and their clinical significance

The suitability of proteome‐based strategies for the targeting of tumor‐associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F‐actin capping protein, γ‐actin, stathmin, tubulin‐α, tubulin‐β and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor‐associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.

Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma.

Heat shock proteins (HSP) are families of highly conserved proteins which are induced in cells and tissues upon exposure to extreme conditions causing acute or chronic stress. They exhibit distinct functions and have been implicated in the pathogenesis of a number of diseases, including cancer. A causal relationship between HSP expression and immunogenicity has been demonstrated in murine and human tumors and is also associated with the immune response. In order to investigate the correlation of HSP expression and their immunogenic potential in renal cell carcinoma (RCC), we here analyzed (i) the protein expression profile of various members of the HSP family in untreated and interferon (IFN)‐γ treated RCC cell lines as well as normal kidney epithelium, and (ii) the anti‐heat shock protein reactivity in sera derived from RCC patients and healthy controls using proteomics‐based techniques. A heterogeneous expression pattern of members of the HSP families was demonstrated in RCC cell lines and in cells representing normal renal epithelium. In some cases the expression rate is moderately altered by IFN‐γ treatment. In addition, a distinct anti‐heat shock protein reactivity could be detected in autologous and allogeneic sera from RCC patients and healthy controls. These data suggest that HSP play a role in the immunogenicity of RCC and thus might be used for the design of immunization strategies to induce a potent antitumor response in this disease.