Quantification of HER Expression and Dimerization in Patients’ Tumor Samples Using Time-Resolved Förster Resonance Energy Transfer

Alexandre Ho-Pun-Cheung,Eric Assenat,Evelyne Lopez-Crapez,André Pèlegrin,Gérard Mathis,Florence Castan,Nadège Gaborit,Patrick Garnero,Caroline Bascoul-Mollevi,Christel Larbouret,Hervé Bazin,Jeanne Ramos,Marc Ychou,Syed A Aziz

doi:10.1371/journal.pone.0037065

Abstract

Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients’ treatment in oncology.

Highlights

The human epidermal growth factor receptor 2 (HER2) is a 185 KDa transmembrane glycoprotein that belongs to the human epidermal receptor (HER) family of receptor tyrosine kinases [1]
To quantify the absolute amount of epidermal growth factor receptor (EGFR) and HER2 using the HER quantification time-resolved Forster resonance energy transfer (TR-Forster resonance energy transfer (FRET)) assays, we used the tumor xenograft models as calibrators to convert the fluorescence signal measured by TR-FRET analysis into the number of receptors per cell (Fig. 2A)
First, we tried to estimate by fluorescenceactivated cell sorting (FACS) the number of receptors per cell in cell suspensions obtained by dissociation of NIH/3T3 EGFR, NIH/3T3 HER2, NIH/3T3 EGFR/HER2 and SKOV-3 tumor xenografts

Summary

Introduction

The human epidermal growth factor receptor 2 (HER2) is a 185 KDa transmembrane glycoprotein that belongs to the human epidermal receptor (HER) family of receptor tyrosine kinases [1]. The United States Food and Drug Administration approved immunohistochemistry (IHC) staining for detecting HER2 protein overexpression and fluorescence in situ hybridization (FISH) assays for quantifying HER2 amplification [14] They are not sufficient for optimal patients’ selection, as less than half of the patients with HER2-positive cancers will respond to trastuzumab therapy [9,10,15]. It is believed that HER2 overexpression causes aberrant activation of intracellular signaling pathways through spontaneous formation of HER2 homodimers and/or increased heterodimerization with other members of the HER family, such as the epidermal growth factor receptor (EGFR) As these receptors can display distinct signaling properties dependent on their dimerization partner [16], quantification of HER dimers could help predicting the patient’s response and outcome to anti-HER therapies. Only few works, if any, have investigated the use of HER dimer expression profile to stratify patients into subgroups who might respond differently to trastuzumab

Methods

Results

Conclusion