Discovery and validation of breast cancer biomarkers using microdissected tissue specimens coupled with targeted and mass spectrometry-based proteomic studies Free

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Introduction
 Mammary carcinoma is the second leading cause of death from cancer among women in western countries. Certain subtypes of mammary carcinoma, i.e. mucinous carcinoma, are particularly notable for their better survival rates when compared to even the lowest grade variety of the more common ductal carcinomas. Mass spectrometry (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 biomarkers for diagnostic and prognostic applications related to breast cancer sub-typing.
 Methods
 One of the main problems with the analysis of tumor tissue specimens 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 protein amounts extracted from targeted tumor cells. To effectively and sensitively analyze minute protein extracts, capillary isotachophoresis (CITP)-based multidimensional separations coupled with tandem MS enables global comparisons of protein expression profiles. These quantitative and comparative proteome studies generate candidate lists for validation using (LC-SRM), and TMA IHC.
 Preliminary Data
 By enriching 10,000 tumor cells from the microenvironment of each tissue specimen subtype, each single proteome analysis yields over 20,000 distinct and high confidence peptide identifications with a false positive rate of less than 1%. These peptide hits lead to the identification of more than 3,500 nonredundant proteins. We have analyzed 5 solid tumor cases, each containing regions of both mucinous and in-situ ductal carcinoma, as well as normal tissue. Several markers have been discovered which distinguish these subtypes with confidence. Two markers have available antibodies which will be used to screen TMAs by IHC to support the capability of the markers to distinguish these subtypes.