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 Yining Shi, PhD, Weidong Huang, MD, Yuping Tan, MS, Xueguang Jin, MS, Rajiv Dua, PhD, Elicia Penuel, PhD, Ali Mukherjee, PhD, Jeff Sperinde, PhD, Herjit Pannu, MD, Ahmed Chenna, PhD, Lisa DeFazio-Eli, PhD, Sailaja Pidaparthi, PhD, Youssouf Badal, PhD, Gerald Wallweber, PhD, Lili Chen, MD, Steve Williams, PhD, Hasan Tahir, PhD, Jeff Larson, PhD, Laurie Goodman, PhD, Jeannette Whitcomb, PhD, Christos Petropoulos, PhD, and John Winslow, PhD 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 immunohisto- chemistry (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 log 10 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 histo- scores 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. Key Words: HER/erbB receptors, HER1, HER2, protein quantitation, receptor dimerization, FFPE tumors, proximity immunoassay, singlet oxygen, capillary electrophoresis, breast cancer (Diagn Mol Pathol 2009;18:11–21) T he epidermal growth factor receptor (EGFR), or HER/erbB family of receptor tyrosine kinases, is widely recognized as a component of signal transduction networks that are dysregulated in several major can- cers. 1–4 EGFR/HER1/erbB1 and HER2/neu/erbB2 are members of the EGFR family, which are genetically amplified or overexpressed in approximately 40% of nonsmall cell lung cancers (NSCLCs) and approximately 30% of breast cancers, respectively. 5–7 Several antibody- based and small molecule-based inhibitors of the HER kinases have been developed as therapeutics and widely tested. Although responses to these agents have been encouraging in some cases, their effectiveness has not been as widespread as predicted. In this respect, only approximately 50% of patients with HER2-positive metastatic breast cancer receiving the anti-HER2 anti- body Herceptin (trastuzumab) derive significant clinical benefit. 8–10 A similar picture has emerged for the prediction of responses to drugs targeted at EGFR in NSCLC. 11–14 There remains a need for new diagnostic tests that are able to identify patient subgroups that may respond to HER1-targeted and HER2-targeted drugs. Currently, 2 commercially available testing methods are used in the clinical evaluation of HER1 and HER2 Copyright r 2009 by Lippincott Williams & Wilkins From the Departments of Research and Development, Clinical Research, and Operations, Monogram Biosciences Inc, South San Francisco, CA. Supported by Monogram Biosciences Inc. Supplemental Figures 1–4 can be viewed online at http://www. molecularpathology.com. Reprints: John W. Winslow, PhD, Research and Development (Oncology), Monogram Bioscience Inc, 345 Oyster Point Boulevard, South San Francisco, CA 94080 (e-mail: jwinslow@monogrambio.com). ORIGINAL ARTICLE Diagn Mol Pathol  Volume 18, Number 1, March 2009 11