Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue.

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry.

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue.

Background: Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies.

Methods: A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform.

Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues.

The heterogeneity of breast cancer requires the discovery of more incisive molecular tools that better define disease progression and prognosis. Proteomic analysis of homogeneous tumor cell populations derived by laser microdissection from formalin-fixed, paraffin-embedded (FFPE) tissues has proven to be a robust strategy for conducting retrospective cancer biomarker investigations. We describe an MS-based analysis of laser microdissected cancerous epithelial cells derived from twenty-five breast cancer patients at defined clinical disease stages with the goal of identifying protein abundance characteristics indicative of disease progression and recurrence.

Application of a global proteomic approach to archival precursor lesions: deleted in malignant brain tumors 1 and tissue transglutaminase 2 are upregulated in pancreatic cancer precursors.

Background: Pancreatic cancer is an almost uniformly fatal disease, and early detection is a critical determinant of improved survival. A variety of noninvasive precursor lesions of pancreatic adenocarcinoma have been identified, which provide a unique opportunity for intervention prior to onset of invasive cancer. Biomarker discovery in precursor lesions has been hampered by the ready availability of fresh specimens, and limited yields of proteins suitable for large scale screening.

Liquid Tissue: proteomic profiling of formalin-fixed tissues.

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.

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).