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  • Title: A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.
    Author: Shi Y, Huang W, Tan Y, Jin X, Dua R, Penuel E, Mukherjee A, Sperinde J, Pannu H, Chenna A, DeFazio-Eli L, Pidaparthi S, Badal Y, Wallweber G, Chen L, Williams S, Tahir H, Larson J, Goodman L, Whitcomb J, Petropoulos C, Winslow J.
    Journal: Diagn Mol Pathol; 2009 Mar; 18(1):11-21. PubMed ID: 19214113.
    Abstract:
    The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.
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