HER2-HER3 dimer quantification by FLIM-FRET predicts breast cancer metastatic relapse independently of HER2 IHC status

Abstract

Overexpression of HER2 is an important prognostic marker, and the only predictive biomarker of response to HER2-targeted therapies in invasive breast cancer. HER2-HER3 dimer has been shown to drive proliferation and tumor progression, and targeting of this dimer with pertuzumab alongside chemotherapy and trastuzumab, has shown significant clinical utility. The purpose of this study was to accurately quantify HER2-HER3 dimerisation in formalin fixed paraffin embedded (FFPE) breast cancer tissue as a novel prognostic biomarker.FFPE tissues were obtained from patients included in the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) study. HER2-HER3 dimerisation was quantified using an improved fluorescence lifetime imaging microscopy (FLIM) histology-based analysis. Analysis of 131 tissue microarray cores demonstrated that the extent of HER2-HER3 dimer formation as measured by Förster Resonance Energy Transfer (FRET) determined through FLIM predicts the likelihood of metastatic relapse up to 10 years after surgery (hazard ratio 3.91 (1.61-9.5), p = 0.003) independently of HER2 expression, in a multivariate model. Interestingly there was no correlation between the level of HER2 protein expressed and HER2-HER3 heterodimer formation. We used a mathematical model that takes into account the complex interactions in a network of all four HER proteins to explain this counterintuitive finding.Future utility of this technique may highlight a group of patients who do not overexpress HER2 protein but are nevertheless dependent on the HER2-HER3 heterodimer as driver of proliferation. This assay could, if validated in a group of patients treated with, for instance pertuzumab, be used as a predictive biomarker to predict for response to such targeted therapies.

Keywords: FLIM-FRET; HER2; HER3; breast cancer; prognosis.

Figure 1. Detection of HER2-HER3 interaction in FFPE cells samples by FRET-FLIM assay

(A) Schema illustrating the principle of antibody-based FRET-FLIM assay. Energy transfer (FRET) occur between donor fluorophore (Alexa546) and acceptor fluorophore (Cy5) upon excitation of donor only at distance less than 10 nm between fluorophores. (B) Interaction between HER2 and HER3 proteins induced by NRG1 treatment. Detected with anti-HER3-IgG-Alexa546 (donor) and anti-HER2-IgG-Cy5 (acceptor) antibodies in FFPE SKBR3 cells. Pseudocolour map shows distribution of measured lifetime where red/yellow pixels represents low lifetime - higher level of HER2-HER3 dimer). Scale bar = 60 μm. (C) Quantification of the result presented in A.

Figure 2. Relationship between HER2-HER3 dimer and expression of proteins in patients' samples

(A) Representative images of tumors with low (upper panels) and high (bottom panels) levels of HER2-3 interaction. (B) Distribution of FRET efficiency signal across patients' tumor cores on Guy's METABRIC TMAs (N = 131). (C) FRET efficiency (mean ± SEM) shown by available HER2 IHC scores (0,1,2,3; HercepTest) and for all imaged cores.

Figure 3. HER2 and HER3 expression levels from IIlumina HT12 microarray

Data are shown for all samples with FRET imaging data for which HER2 status is available by TMA IHC (left: HER2-negative; right: HER2-positive). Points: red, FRET high; black, FRET low. The FRET efficiency threshold to define FRET high/low samples (FRET efficiency = 8.56%) was selected using an exploratory ROC curve analysis to identify an optimal dichotomization (see Methods).

Figure 4. Absence of correlation between level of HER2-HER3 dimer and total HER2, HER3 abundances is revealed by mathematical modelling

(A) Schematic diagram of a simple interaction model between HER1, HER2 and HER3. (B) Schematic diagram of HER1-4 interaction model with possible dimerization events including homo- and hetero-dimerization. The receptors are denoted as E1, E2, E3 and E4 for simplicity in the schemes, species Eij indicates the dimer formed between receptor Ei and Ej (the indexes i and j are between 1 and 4). (C, D) Lack of correlation between steady-state level of the HER2-HER3 dimer and both HER2, HER3 abundances, simulated for 400 simulated patients in the simplified model, when [HER2 total] ~ [HER3 total] << [HER1 total] (C) and [HER2 total] << [HER3 total] << [HER1 total] (D). (E) Lack of correlation between steady-state level of the HER2-HER3 dimer and both HER2, HER3 abundances simulated for 400 simulated patients in the detailed model, when [HER2 total] ~ [HER3 total] << [HER1 total]. Models description is given in the Supplementary Material, Tables S1–S5.

Figure 5. Kaplan-Meier (KM) curves off distant metastasis free survival versus FRET efficiency or HER2 status shown for follow up periods off 5 years (left) and 10 years (right)

(A) KM plots for HER positive and negative tumor samples. (B) KM plots for high and low FRET efficiency.