Abstract A152: Multiplex assay in FFPE tissues to simulateously quantify the human EGF receptor (HER1–4) family proteins: Implications for targeted therapy and resistance to therapy.

Abstract

Abstract The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and Her2), and two receptors (Her3 and Her4) which are the subject of intensive preclinical and early clinical investigation. Although drugs inhibiting both EGFR and Her2 show significant antitumor activity in the clinic, the acquisition of resistance is a hallmark of these and other targeted therapies. In the case of both targets, one of the emerging resistance mechanisms is the co-expression of other members of the EGFR superfamily. It was recently shown that Her2 co-expression mediates resistance in cetuximab treated head and neck cancer (Sci Transl Med 7(3)99). Similarly, much attention has been paid to Her3 both as a bona fide drug target as well as a resistance mechanism. Finally, Her4, though less well studied appears to be another resistance mechanism. Her3 is usually expressed at much lower levels than EGFR and Her2 (often <10%). Yet, receptor crosstalk and heterodimerization can allow even very low levels of Her3 to exert a large impact on drug response. According to one report (Cancer Epidemiol Biomarkers Prev. 19(4):982), IHC analysis of Her3 may be inadequate to properly quantitate Her3 expression in FFPE tumor tissues. This may be a result of high sequence homology/identity between Her3 and other Her family members. Due to the critical need to accurately quantitate this receptor, we have used a mass spectrometric approach to develop a specific and quantitative SRM assay for Her3 from FFPE tumor tissue. By using trypsin digestion mapping, we identified multiple unique peptide sequences from Her3 which were quantitated by SRM Mass spectrometry. Our assay was preclinically validated using the single most sensitive peptide, quantitating Her3 expression in multiple different cell lines, and human NSCLC primary tumor xenografts. These preclinical studies were then extended by assessing the expression levels of Her3 in two cohorts of clinical tumor tissue which had been treated with Her family antagonists. First, we measured Her3 expression in a set of neoadjuvant gefitinib treated NSCLC tumors. In this cohort, 12/15 tumor showed low but measurable levels of Her3 expression, ranging from 50–100 amol/ug tumor tissue. In a second tissue set, we measured Her3 expression in a cohort of advanced (Stage III-IV) breast cancer tissues which had undergone post resection adjuvant treatment with trastuzumab. These breast cancer samples demonstrated a higher level of expression of Her3, ranging from 50 − 250amol/ug tumor tissue, and 15/18 tumors were Her3 positive. In both studies, the relationship between Her family expression and response to either gefitinib or trastuzumab is currently under study. It is critically important to understand mechanisms of resistance in patients undergoing targeted therapies, and Liquid Tissue-SRM promises to be a platform which can deliver extremely high sensitivity, absolute specificity as well as multiplexing capabilities to assess the four HER family targets in unison. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A152.