Discovery and validation of epithelial ovarian carcinoma biomarkers using microdissected tissue specimens coupled with targeted and MS-based proteomic studies
Authors: Weije Wang, Tong Guo, Cheng S. Lee, Paul A. Rudnick, Zhengping Zhaung, Wenxin Zheng, Brian Balgley
Journal: Clinical Cancer Research (2006)

A111   Introduction: Ovarian carcinomas are responsible for 5% of all cancer-related deaths among American women and occur in approximately 2% of the population. Epithelial tumors comprise 80-90% of ovarian carcinomas. These can be differentiated into several subtypes: clear cell, endometriod, mucinous, serous and transitional cell. MS-based proteomic studies have emerged as a powerful tool for biomarker discovery using body fluids, cells, and tissues. However, biomarker discovery efforts have been challenged by the complexity, dynamic range, and quality of proteomes in clinical specimens. Due to the low levels of tumor-associated proteins in serum, a targeted approach, involving the use of clinically well-defined fresh frozen tissues coupled with tissue microdissection, novel proteome technologies and bioinformatics, has been employed to identify and validate unique epithelial ovarian carcinoma biomarkers among the clear cell, endometrioid and serous subtypes for diagnostic and prognostic applications. Methods: One of the main problems with the analysis of tissue samples is the heterogeneous nature of the sample. While methods such as laser capture microdissection enable the isolation of homogeneous subpopulations of diseased cells, proteomic analysis of microdissection-procured specimens is severely constrained by the limited amounts of sample generated. To effectively and sensitively analyze minute protein extracts, capillary isoelectric focusing (CIEF)-based multidimensional separations coupled with ESI-tandem MS enable global comparison of protein expression profiles across various forms of normal and tumor tissues at different stages of disease development. In combination with customized back-end bioinformatic tools and data visualization methodologies, these quantitative and comparative proteome studies generate candidate lists for validation using single reaction monitoring (SRM), immunohistochemistry (IHC), and Western blots.Preliminary Data: Even though microdissection technologies provide enriched, high quality, and reproducible tissue samples, current proteome platforms, including 2-D PAGE and MudPIT, require substantially larger cellular samples which are incompatible with protein extract levels obtained from microdissected tissue specimens. A unique multidimensional separation system, which couples electrokinetic focusing of low abundance proteins with extremely high resolving power of combined CIEF/nano-RPLC separations, greatly enhances the dynamic range and detection sensitivity of MS and enables ultrasensitive analysis of limited tissue samples as demonstrated in our previous studies (Anal Chem, 2005, 77, 6549 and J Prot. Res, 2005, 4, 36). By enriching 10,000 tumor cells from each tissue sample, each single proteome analysis yields over 10,000 distinct and high confidence peptide hits with a false positive rate of less than 1%. These peptide hits lead to the identification of more than 2,500 nonredundant proteins with greater than 80% protein overlapping among repeated runs of the same tissue specimens. To the best of our knowledge, our results present the most comprehensive, high confidence, and reproducible proteome analysis of microdissected tissues. We have performed triplicate analyses of three samples each of three epithelial ovarian cancer subtypes: clear cell, endometrioid and serous. A small set of differentially expressed proteins, as indicated by a t-test score of