Proteomic Analysis of Formalin-fixed Prostate Cancer Tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells. Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells. The ability to correlate results of biomarker discovery investigations to defined disease states promises to improve early detection and provide for targeted therapeutics and markers of drug toxicity. Large scale gene profiling investigations have led to vast amounts of gene expression information on candidate biomarkers, which have proven useful to the understanding of biological and disease processes (1Schena M. Shalon D. Davis R.W. Brown P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Science. 1995; 270: 467-470Google Scholar, 2DeRisi J. Penland L. Brown P.O. Bittner M.L. Meltzer P.S. Ray M. Chen Y. Su Y.A. Trent J.M. Use of a cDNA microarray to analyse gene expression patterns in human cancer.Nat. Genet. 1996; 14: 457-460Google Scholar, 3Krizman D.B. Chuaqui R.F. Meltzer P.S. Trent J.M. Duray P.H. Linehan W.M. Liotta L.A. Emmert-Buck M.R. Construction of a representative cDNA library from prostatic intraepithelial neoplasia.Cancer Res. 1996; 56: 5380-5383Google Scholar, 4Strausberg R.L. Greenhut S.F. Grouse L.H. Schaefer C.F. Buetow K.H. In silico analysis of cancer through the Cancer Genome Anatomy Project.Trends Cell Biol. 2001; 11: S66-S71Abstract Full Text Full Text PDF Scopus (51) Google Scholar). Despite these advances, there is limited information on the gene products that play vital roles in cellular processes including cancer initiation, progression, and metastasis (5Wasinger V.C. Corthals G.L. Proteomic tools for biomedicine.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2002; 771: 33-48Google Scholar). Consequently, a crucial need exists to develop similar large scale approaches for protein biomarker discovery. Tissue-based proteomic studies are inherently attractive for relating protein biomarkers directly to disease. Whereas fresh and/or frozen tissue samples may represent attractive samples from which proteomic biomarker investigations may be conducted, they are often difficult to obtain in large numbers and relatively expensive to store in a stable form. Formalin fixation and paraffin embedding of tissue is the standard processing methodology practiced in pathology laboratories worldwide, resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. Hence, formalin-fixed paraffin-embedded (FFPE) 1The abbreviations used are: FFPE, formalin-fixed paraffin-embedded; IHC, immunohistochemistry; LT, Liquid Tissue™; nanoRPLC, nanoflow reversed-phase liquid chromatography; BPH, prostate hyperplasia; CCD, charge-coupled device; PCa, prostate cancer; PSA, prostate-specific antigen; CAD/CAM, computer-aided design/computer-aided machining; OCT, optimal cutting temperature compound; LIT, linear ion trap. 1The abbreviations used are: FFPE, formalin-fixed paraffin-embedded; IHC, immunohistochemistry; LT, Liquid Tissue™; nanoRPLC, nanoflow reversed-phase liquid chromatography; BPH, prostate hyperplasia; CCD, charge-coupled device; PCa, prostate cancer; PSA, prostate-specific antigen; CAD/CAM, computer-aided design/computer-aided machining; OCT, optimal cutting temperature compound; LIT, linear ion trap. tissues represent a potentially attractive resource for conducting retrospective protein biomarker investigation. Arising from the high degree of covalently cross-linked proteins in FFPE tissue, immunohistochemistry (IHC) is currently the only published technology capable of providing “proteomic” information from these samples, and unfortunately IHC methods lack sensitivity, quantitation, and scalability. Methodologies designed to increase IHC sensitivity by decreasing the effects of formalin-induced protein cross-linking have shown some success, yet quantitation and scalability issues remain (6MacIntyre N. Unmasking antigens for immunohistochemistry.Br. J. Biomed. Sci. 2001; 58: 190-196Google Scholar, 7Shi S.R. Cote R.J. Taylor C.R. Antigen retrieval techniques: Current perspectives.J. Histochem. Cytochem. 2001; 49: 931-937Google Scholar). In addition, IHC requires a priori knowledge of individual proteins being analyzed, thereby limiting biomarker discovery approaches. Other methods to procure and assay soluble proteins directly from FFPE tissue have also shown limited success on a small scale (8Ikeda K. Monden T. Kanoh T. Tsujie M. Izawa H. Haba A. Ohnishi T. Sekimoto M. Tomita N. Shiozaki H. Monden M. Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.J. Histochem. Cytochem. 1998; 46: 397-403Google Scholar, 9Brooks S.A. Dwek M.V. Leathem A.J. Release and analysis of polypeptides and glycopolypeptides from formalin-fixed, paraffin wax-embedded tissue.Histochem. J. 1998; 30: 609-615Google Scholar, 10Izawa H. Yamamoto H. Ikeda M. Fukunaga H. Yasui M. Ikenaga M. Sekimoto M. Monden T. Matsuura N. Monden M. Analysis of cyclin D1 and CDK expression in colonic polyps containing neoplastic foci: A study of proteins extracted from paraffin sections.Oncol. Rep. 2002; 9: 1313-1318Google Scholar). Development of the capability to conduct large scale analyses of tissues using proteomics approaches analogous to the scale and throughput of high throughput gene expression analysis could have far reaching implications on protein biomarker investigations of disease through interrogation of the vast archived FFPE tissue collections. Laser-based tissue microdissection has enabled molecular analysis of specifically defined populations of cells captured directly from their tissue microenvironment (3Krizman D.B. Chuaqui R.F. Meltzer P.S. Trent J.M. Duray P.H. Linehan W.M. Liotta L.A. Emmert-Buck M.R. Construction of a representative cDNA library from prostatic intraepithelial neoplasia.Cancer Res. 1996; 56: 5380-5383Google Scholar, 11Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 274: 998-1001Google Scholar, 12Gillespie J.W. Ahram M. Best C.J. Swalwell J.I. Krizman D.B. Petricoin E.F. Liotta L.A. Emmert-Buck M.R. The role of tissue microdissection in cancer research.Cancer J. 2001; 7: 32-39Google Scholar, 13Knezevic V. Leethanakul C. Bichsel V.E. Worth J.M. Prabhu V.V. Gutkind J.S. Liotta L.A. Munson P.J. Petricoin III, E.F. Krizman D.B. Proteomic profiling of the cancer microenvironment by antibody arrays.Proteomics. 2001; 1: 1271-1278Google Scholar). Despite recent interest in conducting tissue microdissection of FFPE tissues for mRNA extraction and analysis, microdissection of FFPE tissues is not widely practiced due to lack of technological optimization for fixed tissue and lack of methodologies for extraction and analysis of soluble protein (14Cohen C.D. Grone H.J. Grone E.F. Nelson P.J. Schlondorff D. Kretzler M. c-myc antisense oligonucleotide treatment ameliorates murine ARPKD.Kidney Int. 2002; 61: 125-132Google Scholar, 15Gjerdrum L.M. Sorensen B.S. Kjeldsen E. Sorensen F.B. Nexo E. Hamilton-Dutoit S. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma: An alternative method for HER-2/neu analysis.J. Mol. Diagn. 2004; 6: 42-51Google Scholar, 16Gillespie J.W. Best C.J. Bichsel V.E. Cole K.A. Greenhut S.F. Hewitt S.M. Ahram M. Gathright Y.B. Merino M.J. Strausberg R.L. Epstein J.I. Hamilton S.R. Gannot G. Baibakova G.V. Calvert V.S. Flaig M.J. Chuaqui R.F. Herring J.C. Pfeifer J. Petricoin E.F. Linehan W.M. Duray P.H. Bova G.S. Emmert-Buck M.R. Evaluation of non-formalin tissue fixation for molecular profiling studies.Am. J. Pathol. 2002; 160: 449-457Google Scholar, 17Ahram M. Flaig M.J. Gillespie J.W. Duray P.H. Linehan W.M. Ornstein D.K. Niu S. Zhao Y. Petricoin III, E.F. Emmert-Buck M.R. Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications.Proteomics. 2003; 3: 413-421Google Scholar). Dramatic improvements in MS instrumentation and the rapid growth of genomic databases have enabled development of high throughput proteomic approaches to identify and quantify large numbers of proteins from complex samples such as cancer (5Wasinger V.C. Corthals G.L. Proteomic tools for biomedicine.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2002; 771: 33-48Google Scholar, A. M. technology in 2003; Scholar, W.M. J.M. the complementary of and for 2003; 14: Scholar, S.A. protein patterns in disease using mass Res. 2004; 3: Scholar, J. The to 2001; Scholar). of proteins by MS in cells by microdissection of fresh frozen tissue has been L. D. Proteomic analysis of of the breast using capture and Res. 2004; 3: Scholar, H. V. A. Gutkind J.S. analysis of and using microdissection and mass Scholar). The combined of tissue microdissection and MS analysis of FFPE tissue has the for protein biomarker for to the of cancer and have a novel tissue microdissection technique for the extraction of cells from FFPE tissue and a methodology for the of soluble proteins and peptides for analysis by nanoflow reversed-phase liquid for a proteomic of benign prostate hyperplasia and prostate cancer (PCa) The the for of proteins and their expression levels and this information to a disease state from FFPE study the large scale analysis from this study a technological by which the vast archive of pathologically characterized FFPE clinical samples may be utilizing state of the art proteomic acid and were from was from was from and were used as from the and in using a The Liquid protein and the Liquid protein were from A FFPE prostate was and by standard were and on and with and for of tissue analysis was on and were with on the The IHC analysis for prostate-specific antigen was using standard methodologies with a antibody and standard detection tissue tissue were from the FFPE prostate tissue on the and for at was by treatment in for this was by tissue through and was with through and was to microdissection with in for were the and to cellular with which were using the The microdissection was for of the and of the computer-aided design/computer-aided was by to at a and throughput the defined and cellular to of cells the a The tissue microdissection is on designed to a a a tissue An at the was for are of with with the of a standard The is a with a of The is by a to and the to and to a which the the and for of the a the of formalin fixation on the ability to identify peptides extracted from FFPE tissue a was from a A of the was of the was frozen on and at to embedding in optimal cutting temperature and The was in formalin for to embedding in paraffin and The frozen tissue were in for and for to the OCT, by in and and The FFPE tissue was in of for by through and The from the formalin-fixed and frozen tissues were in of and by as by microdissection for analysis and analysis were by to the for analysis was in of at for on for at which of B was by at for were stored at for analysis was in of at for on for at which of was by at was to a of and the samples were for at to were stored at of protein were in on a using a A protein assay for expression was using standard with a antibody and a antibody phosphatase and for of the were captured by a as a and with analysis for The were a for and analysis peptides from BPH and PCa cells were using to and in for the isotope protein from of microdissected cells were and in and (PCa) containing was in the and to at of were for at which of the was and the samples were for peptides are the extraction of the proteins from the cells from the FFPE the role of this is to the with the was to a of and the were for were to and in to was using to a linear ion mass a of were using that were with of the was for with A acid in at a of were using a linear of B acid in to B in to B in at a of The was in a in which MS is by the molecular are for using a of was to of peptides for The temperature and were at and were a mass to ion of by in the using the and for the was using detection in for the MS at by using the on the molecular in the mass were the human and proteomic databases from the using were using and a on the for the a to be identified, to state and of for for and for for the and a of results were using developed to peptides and the PCa was a human protein in a similar as J. Evaluation of with mass for protein The Res. 2003; Scholar). The that using the results in of the in being as a The molecular and cellular of the proteins in the analyses in this study were by gene for protein were to the method of and J. R.F. A method for the of a Mol. Biol. Scholar). A FFPE prostate tissue from a was for this tissue a of and of prostatic intraepithelial and analysis of this prostate prostatic and and and and of prostatic intraepithelial hyperplasia and (6MacIntyre N. Unmasking antigens for immunohistochemistry.Br. J. Biomed. Sci. 2001; 58: 190-196Google the ability to extract protein from FFPE tissue, IHC was to identify tissue that and tissue from the prostate tissue PCa and PCa BPH and and a The IHC analysis that cells and the tissue for expression including the PCa and and BPH and the prostate tissue were and on for cells from of the and cells from of the BPH and and cells from the were microdissected using a novel tissue microdissection technique developed specifically for FFPE tissue, the The the and tissue a a of tissue from the a The of tissue in a is a of the of the the tissue and the The the that the tissue was to microdissection of cellular for Laser were at a for of cells in a A in with the for tissue of populations for and of the The of microdissected cells was by the of the for the of a cancer and of the were for microarray analysis from the PCa and and on a microarray in with of protein were at the high of the detection of the protein with a antibody detection of in the from the cancer whereas was in the from the shown in the ability to expression in a microarray and the of extracted protein from formalin-fixed A antibody not samples were for protein the of proteomic methods for conducting of peptides extracted from FFPE PCa tissue, cells from PCa and BPH A and were microdissected as and by of protein from extract were by resulting in the of and peptides and from the BPH and PCa FFPE tissue and these were by peptides in tissue A and many of the proteins by the of peptides represent highly proteins A of proteins were in this as in microdissected PCa tissue extract including PSA, prostatic acid phosphatase macrophage inhibitory cytokine-1 and protein of these markers were by peptides in the protein from PCa and BPH cells. was by and peptides in the protein from the PCa and BPH cells, whereas was by peptides from the PCa microdissection and peptides from the In growth known as was by peptides in the protein extracted from the PCa cells, whereas peptides to were in the protein extracted from the BPH mass of peptides from PCa tissue extract for PSA, and Large An peptides extracted from FFPE tissues is the of resulting from formalin fixation and for resulting from formalin a of the analyses of the FFPE PCa cells were for a arising from formalin fixation. The results that of the peptides the of formalin fixation and is not a of the to be An of the of that of the the peptides in the analysis of this PCa were In a published analysis of peptides that a of of peptides were found to be J. Evaluation of with mass for protein The Res. 2003; Scholar). The results that formalin fixation and not in degree of The results of this study the effects of formalin fixation on tissue biological in this study are on the and quantitation of formalin fixation the numbers of peptides from the PCa tissue analysis with and were The results that the of peptides with is and that the of these proteins is of the would have been a of peptides containing and the results not the they not to a that formalin fixation not have a on the of The of peptides that was also of the peptides this is at the high this may be by the that of the proteins may not be and some and are not to The molecular and cellular of the proteins in the analysis of the FFPE PCa and BPH tissue samples were using gene as shown in The proteins were found to have a of molecular and from The gene of the proteins in these samples was compared with the proteins the human protein analysis that there is in the of proteins from FFPE Whereas the human is to a of proteins in the from the proteomic analysis of FFPE PCa and BPH tissue, this is not the methodology for protein In the of the analysis of the FFPE tissues a that the human is to however, that proteins are to be and would be that they would be in this analysis compared with their in the human The of for the proteins from the PCa and BPH tissues was also J. R.F. A method for the of a Mol. Biol. Scholar). The proteins in similar results and the of to A of the proteins are in of to that for the human protein not In these results are with proteomic analyses in T. Y. J. H.J. analysis of the Cell 2004; 3: Scholar, G. J. H.J. A. Quantitative analysis of the molecular using stable isotope labeling in a Scholar). L. M. M. T. G. D. A. A. T. T. D. and D. A of the that a analysis of FFPE tissue samples utilizing the methodology results in a of protein and In to the to and as isotope labeling was to protein expression microdissected BPH and PCa tissue from a of microdissected cells A and from pathologically prostate tissue and PCa were and The BPH extract was in a in and the PCa extract was in the in The of to these samples the of of at the of for of in the in in the of results in a increase in mass of compared with peptides in A. J. C. labeling for studies with of 2001; Scholar, M. of and its for protein by and mass 1996; Scholar, K. A mass technique a and and a that J. Scholar). of the proteins in the analysis were in the samples, including PSA, and prostate proteins was with levels in tissue on the isotope whereas only a from PCa was An analysis the of that be from of fresh frozen and FFPE cells was to the of formalin cross-linking and paraffin embedding on extraction tissue was of the was frozen and in the was formalin-fixed and A tissue was from the of and cells were extracted from and using the of the analysis of the peptides extracted from the FFPE and frozen are shown in A and similar A of is also shown in A and These the in the with to as as this analysis, peptides to proteins were from the frozen tissue whereas peptides to proteins were from the FFPE tissue A gene of the proteins similar protein and in tissues A of the proteins on the of from the FFPE tissue with the proteins from the frozen tissue in samples a protein that is to human was by far the protein with and peptides from the FFPE and frozen tissue, that the potentially from the FFPE on the extraction of a of proteomic information be extracted from these samples as compared with frozen tissue of the of peptides and proteins from FFPE and frozen of of in a the cellular and molecular of the proteins extracted from FFPE and fresh frozen Large of the of peptides for the proteins the FFPE tissues with the of peptides for protein extracted from frozen of protein in a A analysis of FFPE prostate tissue has been resulting in the of of proteins in of the tissue The results the ability of a protein methodology to enable proteomic investigations of microdissected FFPE A of proteins were in this as in microdissected PCa tissue extract including PSA, and which is to be in benign and prostate growth J. of human prostatic acid phosphatase and the growth of prostate Res. has been used as a for PCa has sensitivity and N. E. antigen as a for of the J. Scholar, acid phosphatase in limited clinical Scholar). is a of the growth of proteins that play a role in and to M. C. A. K. of a novel of the Res. Scholar, H. Z. Brown D. inhibitory A novel biomarker for Cancer 2003; Scholar). has been shown that is highly in PCa tissue and may play a role in the that to PCa J. K.A. M. J. H. J. in cytokine-1 gene with prostate Cancer 2004; Scholar). as is in a of and has been to have and processes H. Z. Brown D. inhibitory A novel biomarker for Cancer 2003; Scholar, J. K.A. M. J. H. J. in cytokine-1 gene with prostate Cancer 2004; Scholar). studies have that protein levels of in prostate tissue cellular and results in Z. M. The role of protein in and 2004; Scholar, Z. M. The of a prostate cancer metastasis Cell Scholar). Analysis of the tissue extract similar results with a A of the prostatic markers in the cancer extract were by similar numbers of peptides in the BPH was by in BPH, and IHC the of in cancer and BPH from the tissue is not has been for its to the prostatic is by the quantitative proteomic analysis utilizing isotope labeling peptides were with expression levels in tissue on the isotope In the of peptides were in the tissue peptides were in the tissue is in PCa cells lack of protein expression has been with BPH Y. T. of macrophage inhibitory in benign prostatic 2004; whereas high levels of expression have been to early prostate H. M. D. T. M. J. profiling of microdissected prostate tissue growth to prostate Res. 2004; Scholar). is by the quantitative proteomic analysis expression was in the cancer extract by whereas expression was in the BPH of expression in this of In addition, a proteins were as PCa and BPH by FFPE tissues are a largely archive in is to the effects of formalin fixation and on the is difficult to identify a complex however, that of the peptides a and the of peptides containing is not that in proteomic studies of fresh cells J. Evaluation of with mass for protein The Res. 2003; Scholar, T. Y. J. H.J. analysis of the Cell 2004; 3: Scholar). not of the that may be in proteins extracted from FFPE biological information be from the analysis of The ability to conduct proteomic investigations of FFPE tissue has the to provide information disease states and may in the development of clinical and/or the study of FFPE tissues has been limited to analysis of protein and expression through of such as and in The methodology large scale proteomic such as to be to vast of clinical and tissues that been of as to such Use of tissue microdissection the ability to correlate protein expression to information for of a of proteins and their in disease. In to tissue microdissection the novel microdissection technology has been developed and for of cells directly from standard formalin-fixed tissue for protein In addition, this technology is a method that cells directly a providing the of for high throughput tissue microdissection The of prostate cancer is using and is to from using a of the of protein expression in with may for BPH and PCa, and with