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.