Quantitative Fluorescence Imaging Analysis for Cancer Biomarker Discovery: Application to B-Catenin in Archived Prostate Specimens Dali Huang, 1 George P. Casale, 1 Jun Tian, 1 Nizar K. Wehbi, 1 Neil A. Abrahams, 2 Zahid Kaleem, 4 Lynette M. Smith, 3 Sonny L. Johansson, 2 Johny E. Elkahwaji, 1 and George P. Hemstreet III 1 1 Department of Surgery, Urologic Surgery Section; Departments of 2 Pathology and Microbiology, and 3 Preventive and Societal Medicine, University of Nebraska Medical Center, Omaha, Nebraska; and 4 Department of Pathology, Veterans Affairs Medical Center, Omaha, Nebraska Abstract The surprising disparity between the number of protein- encoding genes (f30,000) in the human genome and the number of proteins (f300,000) in the human proteome has inspired the development of translational proteomics aimed at protein expression profiling of disease states. Translation- al proteomics, which offers the promise of early disease detection and individualized therapy, requires new methods for the analysis of clinical specimens. We have developed quantitative flourescence imaging analysis (QFIA) for accu- rate, reproducible quantification of proteins in slide- mounted tissues. The method has been validated for the analysis of B-catenin in archived prostate specimens fixed in formalin. QFIA takes advantage of the linearity of fluores- cence antibody signaling for tissue epitope content, a feature validated for B-catenin in methacarn-fixed prostate speci- mens analyzed by reverse-phase protein array analysis and QFIA (r = 0.97). QFIA of B-catenin in formaldehyde-fixed tissues correlated directly with B-catenin content (r = 0.86). Application of QFIA in a cross-sectional study of biopsies from 42 prostate cancer (PC) cases and 42 matched controls identified B-catenin as a potential field marker for PC. Receiver operating characteristic plots revealed that B- catenin expression in the normal-appearing acini of cancer- ous glands identified 42% (95% confidence intervals, 26-57%) of cancer cases, with 88% (95% confidence intervals, 80-96%) specificity. The marker may contribute to a PC biomarker panel. In conclusion, we report the development and validation of a new method for fluorescence quantification of proteins in archived tissues and its application to archived specimens for an evaluation of B-catenin expression as a biomarker for PC. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1371–81) Introduction The availability of structural and functional data for thousands of cellular proteins (1) has inspired widespread interest in translational studies aimed at protein expression profiling of clinical diseases and their antecedent states. A key to the implementation of this translational research is the develop- ment of techniques that offer sensitive, continuous quantifica- tion of proteins in clinical specimens. Gene microarray studies have stimulated the development of protein profiling techni- ques such as protein microarray analysis (2), biological mass spectrometry (3), and tissue-based fluorescence analysis (4, 5), which are applied to small tissue samples, e.g., biopsies. The application of gene microarrays to molecular profiling of cancers provided the first glimpses into the remarkable molecular heterogeneity of cancer among patients and among tumors in the same patient, and identified cancer subclasses defined by common expression patterns (6). In relation to cancer protein profiling, gene microarrays have identified numerous proteins, the expressions of which may be modified (7-10). Protein networks directly carry out cellular functions, and dysregulation of these networks via altered protein expression or posttranslational modification underlies disease. Reverse-phase protein array analysis (RPPA; refs. 2, 6, 11-13) and automated quantitative analysis (AQUA; refs. 4, 5, 14) are two recently introduced methodologies that precisely quantify proteins and their secondary modifications in clinical speci- mens, i.e., slide-mounted tissues. RPPA was developed with a focus on modifications of cell-signaling cascades in disease, and simultaneously analyzes multiple lysates of specimens from one or more patients. Nanoliter volumes of lysate are arrayed onto nitrocellulose slides and probed with a primary antibody detected with a fluorophore-conjugated secondary antibody. Protein array analysis exhibits high sensitivity, precision, and linearity across a wide range of protein concentrations. AQUA was developed with a primary focus on rapid and precise quantification of protein expression in tissue microarrays (TMA)forlarge-scalestudiesofdiseaseoutcome.AQUAentails mapping subcellular compartments of tissue specimens by fluorescence antibody labeling, and assignment of the fluores- cence signal from biomarkers of interest to specific compart- ments. The assay has produced remarkable improvements overpathologistclassificationofimmunohistochemicalstaining in the prediction of population-based disease outcomes. The present study introduces a novel quantitative fluorescence imaging analysis (QFIA) procedure for the rapid and precise quantification of proteins and their secondary modifications in the context of conventional histologic features of slide speci- mens. The procedure complements RPPA and AQUA. Diagnosis of prostate cancer (PC), the second leading cause of cancer deaths among U.S. men (15), is dependent on the quantification of serum prostate-specific antigen. A prostate- specific antigen test of 4 to 10 ng/mL, however, has a specificity of only 25% (16), and requires biopsy, which has 1371 Cancer Epidemiol Biomarkers Prev 2007;16(7). July 2007 Received 8/23/06; revised 3/12/07; accepted 5/2/07. Grant support: Department of Defense Idea Development Award no. DAMD17-02-1-0121, 2002 to 2005, by a National Cancer Institute Cancer Center Support grant P30 CA36727, and a Nebraska Department of Health Institutional LB595 grant for Cancer and Smoking Disease Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: D. Huang and G.P. Casale have contributed equally to the production of this manuscript. Requests for reprints: George P. Hemstreet III, Department of Surgery, Urologic Surgery Section, 982360 Nebraska Medical Center, Omaha, NE 68198-2360. Phone: 402-559-4684; Fax: 402-559-6529. E-mail: gphemstreet@unmc.edu Copyright D 2007 American Association for Cancer Research. doi:10.1158/1055-9965.EPI-06-0718 on June 7, 2020. © 2007 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from