August 1, 2008 (Vol. 28, No. 14)

Precisely Determining How Expression Levels Correspond with Drug Action

Formalin fixed paraffin-embedded (FFPE) tissue constitutes an enormous untapped resource for discovery, validation, and measurement of valuable biomarkers of disease progression, drug response, and toxicity. Millions of FFPE samples are routinely collected and stored in medical and research facilities each year along with patient medical histories and outcomes. Although formalin fixation creates chemical cross-links that stabilize the proteins, until recently protein analysis on these samples was limited to standard immunohistochemistry (IHC).

Expression Pathology has developed tissue proteomic tools to study proteins in FFPE samples. EPI’s Liquid Tissue® sample-preparation and Director™ laser microdissection technologies enable detailed mass spectrometry (MS) analysis and measurement of proteins in FFPE tissue samples (Figure 1).

The Liquid Tissue MS Protein Prep Kit completely solublizes and captures the entire protein content from microdissected FFPE tissue. The protocol is optimized to process 30,000 cells—the equivalent of a 2 x 4 mm region from a 10 µm thick section. It yields 4–7 µg of total protein, which is sufficient for multiple MS analyses.

Director laser microdissection slides, engineered for use with existing laser microdissection systems, provide a fast, easy, and accurate way to collect specific cellular features from FFPE sections.

The availability of these tissue tools has resulted in a growing number of researchers reporting successful identification of novel differentially expressed proteins in a wide range of tissue types and disease conditions.

Lung Cancer Metastasis Biomarkers

Tokyo Medical University has combined global proteomic profiling to identify candidate protein biomarkers with multiple reaction monitoring (MRM) quantitative analysis for validation (Figure 1). A retrospective proteomic analysis of FFPE tissues was undertaken to determine molecular profiles of non-small-cell lung cancer stages IA and IIIA and to assess key proteins relating to premetastatic niches in mediastinal lymph nodes.

Tumorigenic epithelial cells were collected from patient tissue by Director laser microdissection. Proteins were extracted with Liquid Tissue reagents and subjected to LC/MS/MS proteomic profiling. Spectral data was processed by label-free semiquantitative comparison with both spectral counting and ion signal-based statistical approaches, resulting in the identification of 40 molecules distinguishing IA and IIIA stages.

Candidate molecules of interest were further analyzed using MRM mass spectrometry. Results showed that, among other things:

>Seven secretory proteins were upregulated in IIIA stage tumors: S100A9, A100A8, secreted cement gland protein XAG-2 homolog (AGR2), dermcidin, neutophil defensin 1, mesotrypsinogen, and a-1 protease inhibitor,

>S100A9 increased expression in metastatic lymph nodes,

>Napsin-A expression decreased considerably in a subset of stage IIIA patients known to have deceased within three years post surgery,

>Expression levels of NAPSA in primary tumors of stage IIIA lung cancer are prognostic for survival or death within 24 months post diagnosis.

Figure 1

Breast Cancer Biomarker Expression

IHC is the current standard method for protein analysis of FFPE tissue, employing antibodies that bind specifically to antigens in the tissue. IHC is time and labor intensive with little multiplexing capabilities and requires visual interpretation and scoring of stained tissue. False negatives can result from unsuitable antibodies, an inaccessible protein target, or low protein levels. The Liquid Tissue MS approach has many potential advantages including speed, cost, sensitivity, and accurate quantitation.

HER2/neu is notable for its role in the pathogenesis of breast cancer and as a target for treatment. A project to demonstrate the ability of the Liquid Tissue MS approach to provide for quantitative measure of this clinically relevant protein in FFPE tissue was undertaken with NextGen Sciences. This project applied an antibody-free multiple reaction monitoring approach to determine absolute quantitation of HER2 expression in FFPE tissue excised from breast cancer patients.

After microdissection and Liquid Tissue preparation of cancerous breast epithelium, a labeled synthetic peptide specific to the HER2 protein was spiked into each preparation to serve as an assay standard. Samples were injected directly into a triple quadrapole MS and analyzed by MRM.

By comparison to the spiked standard, accurate quantitation was achieved for expression of HER2 protein. Results were compared to IHC and FISH analysis of the same samples, representing the entire range of HER2 expression levels. Results from the combined MRM, IHC, and FISH analyses of HER2 expression were compared (Figure 2).

The MRM assay provides quantitative levels of the HER2 protein in FFPE tissue that correlate well with existing methods but with a wider dynamic range, providing greater discrimination than IHC and FISH scoring. Compared to FISH and ICH, this method provides numerical quantitation and has the potential for faster, less expensive, more sensitive assays.

The Liquid Tissue HER2 MRM approach represents the first time absolute quantitation of protein expression has been demonstrated in FFPE tissue samples. This ability is of immediate interest in preclinical studies and clinical trials to determine how quantitative protein expression levels correlate with drug action or toxicological effects.

Figure 2

Casey Eitner is president and CEO, and David B. Krizman, Ph.D. ([email protected]), is CSO at Expression Pathology.
Web: Phone: (301) 977-3654.

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