Quantification of HER1, HER2 and HER3 by time-resolved F\xf6rster resonance energy transfer in FFPE triple-negative breast cancer samples

Alexandre Ho-Pun-Cheung,André Pèlegrin,Jean-Pierre Bleuse,Hervé Bazin,William Jacot,Evelyne Lopez-Crapez,Thierry Chardès,Joëlle Simony-Lafontaine,Emeline Landas,Gérard Mathis,Florence Boissière-Michot,Jean-François Prost,Caroline Mollevi

doi:10.1038/s41416-019-0670-8

Abstract

BackgroundTriple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence.MethodsWe developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival.ResultsThe assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003).ConclusionsOur TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.

Highlights

Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed
As dysregulated expression of human epidermal receptor (HER) family members is frequent in breast cancer, and given their crucial role in proliferation,[9] these receptors have been extensively investigated as targets for anticancer therapy, HER1, human epidermal growth factor receptor 2 (HER2) and HER3
The median coefficient of variation values between technical replicates were 5.3% (HER1), 5.2% (HER2), and 8.2% (HER3). These results demonstrate that the Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays allow the reliable and precise quantification of HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour samples

Summary

BACKGROUND

The term triple-negative breast cancers (TNBC) was first used in 20051 to describe a subset of tumours characterised by absence or low levels of expression of oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2. IHC typically relies on chromogenic detection that has a narrow linear dynamic range, limiting its ability to generate accurate quantitative results.[19] IHC scoring system is subjective and this can lead to reproducibility issues.[20,21] to improve the accuracy and reproducibility of HER family member quantification in tumours, we developed a timeresolved Förster resonance energy transfer (TR-FRET) method that allows the quantitative and objective measurements of protein expression levels This method relies on the energy transfer between two fluorophores, a donor and an acceptor.[22] When the donor (in this case, a lanthanide complex characterised by a long fluorescence lifetime) is excited by an energy source, it transfers its excitation energy to the acceptor only if the two fluorophores are in close proximity. We evaluated HER quantification by TR-FRET assays and assessed the prognostic role of HER1, HER2 and HER3 expression in TNBC

METHODS

Findings

DISCUSSION