Monica Brown Jones 1 Henry Krutzsch 1 Hungjun Shu 2 Yingming Zhao 2 Lance A. Liotta 1 Elise C. Kohn 1 Emmanuel F. Petricoin III 3 1 FDA-NCI Clinical Proteomics Program, Laboratory of Pathology, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA 2 Department of Biochemistry and Pharmacology, University of Texas, Southwestern Medical School, Dallas, TX, USA 3 FDA-NCI Clinical Proteomics Program, Division of Therapeutic Products, Center for Biologic Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA-125, the cur- rently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI-FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two-dimensional gel electrophoresis (2-D PAGE) to identify proteins that may serve as invasive ovarian cancer-specific biomarkers for early detection and/or new therapeutic targets. We performed 2-D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two non- invasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2-D gels created from microdissected lysates with SYPRO-Ruby stained 2-D PAGE profiles of the patient-matched undissected bulk tumor lysates from all five patients. Twenty-three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or under- expressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and pre- liminary target validation of the mass spectrometry identified proteins cut from the Ruby-red stained gels was performed by LCM coupled Western blot and reverse- phase array technology in a study set of six cases (the aforementioned five cases used in the 2-D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G-protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely over- expressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype. Keywords: Ovarian / Microdissection / Cancer / Biomarkers / Laser capture PRO 0166 1 Introduction The development of a tumor marker specific for epithelial ovarian cancer or new therapeutic targets has been chal- lenged by the limited resource of normal ovarian tissues as comparators, the use of ovarian cancer cell lines that may lack biological relevancy, and the identification of proteins derived from heterogeneous population of cells derived from homogenized whole tissue sections. Laser capture microdissection (LCM) and 2-D PAGE analysis has provided the technical advances to overcome some of the above limitations, and has recently been applied successfully to the analysis and characterization of changes in protein expression in breast, prostate, esoph- ageal cancer, and cervical cancers [1–4]. In these studies, it was repeatedly demonstrated that LCM generated 2-D PAGE profiles showed dramatic enrichment of disease- specific protein signature spots over undissected whole cell lysates, were dramatically different than the prote- omic fingerprints from patient-matched cell lines, and showed no demonstrable laser-induced damage to the LCM- procured proteins. LCM was developed in our laboratory as a method of selective cell procurement and consists of a standard inverted microscope, connected to a low energy (around infrared spectra) laser [5, 6]. After Correspondence: Dr. Emanuel F. Petricoin III, Bldg. 29A Room 2B02, 8800 Rockville Pike, Bethesda, MD 20892, USA E-mail: petricoin@cber.fda.gov Fax: +1-301-408-3256 Abbreviations: LCM, laser capture microdissection; LMP , low- malignant potential; RhoGDI, Rho G-protein dissociation inhibi- tor 76 Proteomics 2002, 2, 76–84 ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 1615-9853/02/0101–76 $17.50+.50/0