Formalin-fixed paraffin-embedded (FFPE) tissue banks are a huge, untapped resource available for proteomic biomarker analysis. They are suitable source material for current proteome profiling techniques such as liquid chromatography–tandem mass spectrometry (LC-MS/MS). In comparison with traditional immunological methods, mass spectrometric proteome analysis offers a more efficient high-volume, multi-analyte method for biomarker discovery and identification of new therapeutic targets.
While the methodology for frozen specimens is not consistent, tissue handling protocols for FFPE are well established. Although mixed cell populations are present in FFPE samples, cell architecture is well preserved; serial slices present opportunity for subsequent immunohistochemistry and mRNA evaluation.
Paulo et al. (2013) compared proteomes from three distinct exocrine pancreatic pathologies — chronic pancreatitis, autoimmune pancreatitis and pancreatic cancer — using LC-MS/MS techniques to analyze the banked FFPE specimens.1 Proteins were extracted from uniform amounts of the FFPE tissue using standard methods. Peptides were isolated using C18 PepClean spin columns (Thermo Scientific) before separation by reverse-phase high-performance liquid chromatography (Thermo Scientific). Eluted peptides were then analyzed using an LTQ FT Ultra mass spectrometer (Thermo Scientific).
In total, nine FFPE specimens were examined, with tissue from three individual patients representing each pathology. A total of 386 non-redundant proteins were discovered by MS analysis from all tissue samples, with 211, 265 and 280 found in autoimmune pancreatitis, chronic pancreatitis and pancreatic cancer, respectively. Of these, 139 proteins were common to all three pathologies.
Further analysis of the differential proteome profiles in each disease cohort showed that 29 proteins were found exclusively in autoimmune pancreatitis tissue samples, 53 in chronic pancreatitis and 73 in pancreatic cancer. The authors note that these proteins warrant further characterization and investigation as potential biomarkers. This finding is of great interest in differentially diagnosing autoimmune pancreatitis from pancreatic cancer. Although each condition differs in prognosis and treatment, both can present similar clinical symptoms and comparable tissue biopsies, thus complicating treatment protocols.
Paulo et al. show the potential for using archived FFPE tissues in proteomic research, noting that despite the potential interference by formalin-induced cross-linking, proteins were reliably extracted from all samples.1 Moreover, they present data showing individual proteins exclusive to each of the exocrine pancreatic pathologies studied. The researchers have shown that banked FFPE specimens are a viable source of information and of great relevance in the future development of novel therapeutic strategies and investigation of diagnostic biomarkers.
1. Paulo, J.A., et al. (2013) “A proteomic comparison of formalin-fixed paraffin-embedded pancreatic tissue from autoimmune pancreatitis, chronic pancreatitis, and pancreatic cancer,” Journal of the Pancreas, 14(4) (pp. 405–414).
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