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Proximal Tissue Fluids and Targeted Proteomics: A Novel Approach to Identify Aggressive Prostate Cancers
The ability to predict which patients have aggressive prostate cancer, warranting a radical course of treatment, from patients with slow-growing tumours that would be better suited for active surveillance, is a major challenge in personalized care for prostate cancer patients. Further, as the incidence of prostate cancer rises, more and more patients are being over-diagnosed, partly due to a lack of reliable prognostic biomarkers. A successful biomarker (or a panel of them) would, in a timely manner, clearly demarcate these patient groups.
In the current article, we describe our approach to biomarker discover utilizing mass spectrometry for the identification of potential soluble protein markers in expressed prostatic secretions (EPS).[1] As a result, we identified several putative prostate cancer biomarkers, demonstrating their differential expression between patient groups with extracapsular and organ-confined tumours, as well as recurrent versus non-recurrent individuals. Furthermore, in a proof-of-concept study, we developed mass spectrometry-based quantitative assays for several candidates and verified them in clinically stratified pools of urinary EPS. This analysis has resulted in the selection of over 100 protein biomarker candidates that warrant verification in large cohorts of clinical samples.
In recent years, large international genomics initiatives (The Cancer Genome Atlas and The International Cancer Genome Consortium) have been assembled to investigate genomic alterations in various cancers. The main goals of these consortia are to identify genomics-based drivers in these cancers using next-generation sequencing platforms. Although these data have provided unprecedented insight into the genomic landscape of cancer, the results are not readily transferable to clinical tests using available body fluids. In addition, genomic information often fails to correlate with protein expression. Our lab is focused upon the application of protein-based mass spectrometry, termed proteomics, for the identification, and quantitative measurements of proteins in complex samples (i.e. tissue, body fluids). Most discovery proteomics platforms enable researchers to rapidly identify thousands of proteins in a single biological sample, thus providing a fingerprint of the physiological state of the tissue or body fluid. Following this discovery, putative candidates are selected for targeted mass spectrometry, enabling multiplexed, and highly quantitative measurements of dozens of proteins in a high-throughput fashion.
The use of body fluids is an attractive starting point for the discovery of biomarkers that can be derived from minimally invasive and accessible sources. The latest generation of mass spectrometry instruments can achieve unsurpassed depth, but discovery proteomics efforts in blood (i.e. serum and plasma) are still plagued by the large dynamic range of proteins and subsequent dilution of low-abundance, albeit potentially interesting, markers. Our group has developed proteomics strategies for the analysis of tissue-proximal fluids [1, 2, 3] with the rationale that capturing proteins that are shed or secreted by the organ of interest into the immediate microenvironment may increase their chances of being identified. With the recent FDA approval of the prostate cancer antigen 3 (PCA-3) test, standardized bio-banking of EPS-urine (a prostate proximal fluid) is already increasing in frequency, making biomarker validation efforts in this clinically applicable fluid more attractive.
Advanced technologies generate long lists of cancer biomarker candidates, but few have found their way into clinical practice. A major bottleneck in the road from discovery to clinical use is the validation phase. Affinity-based assays, such as enzyme-linked immuno/sorbent assays (ELISA), are commonly used for this purpose but they rely on antibodies, which are often not readily available. The process of developing and validating an ELISA assay is slow and expensive, and thus large-scale development, which is needed to sift through long lists of candidates, is unrealistic at this point in time. ELISA assays are also difficult to multiplex, due to high background and non-specific signals as a result of antibody cross-reactivities.
Selected reaction monitoring mass spectrometry (SRM-MS) is a tool that can be used to quantify proteins directly in biological samples. Unlike ELISA, which utilizes antibodies that recognize, presumably, a single epitope on its antigen, SRM-MS relies on the unique biophysical properties of each peptide analyzed. This allows us to target and measure its quantity amongst hundreds of thousands of other peptides from highly complex backgrounds. Importantly, SRM-MS has high-throughput capability; hundreds of peptides can be multiplexed into a single mass spectrometry run, and once an assay has been established for a given protein, it can be used in any sample type, across different mass spectrometry platforms. Therefore, this method offers a rapid and economical pre-validation step of high numbers of biomarker candidates, prior to the generation of the more traditional ELISA assays.
In our proof-of-concept study, we demonstrate the use of multiplexed SRM-MS assays for measuring a number of candidates in EPS-urine. The next steps we take will be dedicated to expanding this approach to a large number of biomarker candidates in individual patient samples. We anticipate the establishment of a panel of protein biomarkers, directly quantifiable and verified in this fluid. We are also interested in exploring EPS subproteomes, particularly the exosomal and glycoproteome, for an in-depth view of this complex fluid. However, future studies are not limited to this - our updated EPS database contains over 1 200 proteins, and this information-rich dataset has already shown us that certain pathways are enriched in different stages of disease. Further investigation can expand our knowledge of prostate cancer progression and uncover therapeutic targets for improved therapy.
References:
- Kim, Y., Ignatchenko, V., Yao, C. Q., Kalatskaya, I., et al., Identification of Differentially Expressed Proteins in Direct Expressed Prostatic Secretions of Men with Organ-confined Versus Extracapsular Prostate Cancer. Mol Cell Proteomics 2012, 11, 1870-1884.
- Drake, R. R., Elschenbroich, S., Lopez-Perez, O., Kim, Y., et al., In-depth proteomic analyses of direct expressed prostatic secretions. J Proteome Res 2010, 9, 2109-2116.
- Principe, S., Kim, Y., Fontana, S., Ignatchenko, V., et al., Identification of prostate-enriched proteins by in-depth proteomic analyses of expressed prostatic secretions in urine. J Proteome Res 2012, 11, 2386-2396.
Written by:
Yunee Kim, Raymond Lance, Richard R. Drake, O. John Semmes, and Thomas Kislinger as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
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