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Characterisation of HER heterodimers in breast cancer using in situ proximity ligation assay

  • Preclinical Study
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Abstract

HER2 plays an important role in breast cancer progression and provides predictive and prognostic information. HER2 receptor family members function through dimerisation, which can lead to impact on cell function, growth and differentiation; however, their value in breast cancer development remains to be defined. This study aims to examine the relationships of HER2 heterodimers to breast cancer characteristics in trastuzumab naïve and treated cases. HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER2 heterodimerisation status [chromogenic in situ proximity ligation assay (PLA)] were assessed in two breast cancer series prepared in tissue microarray (TMA) format. A range of signals/cell for each HER2 heterodimer was detected (0–34.6 signals/cell). The vast majority of cases with HER2 heterodimers showed HER2 gene amplification and/or protein expression. There was an association between HER2 dimerisation with HER3 and HER4 and their protein expression level but no such association was found in with HER1 (EGFR). Of the HER2+ cases, 74, 66, and 58 % showed heterodimers with EGFR, HER3 and HER4, respectively. 51 % of HER2+ tumours expressed all three heterodimers whereas 23 % of the cases did not show expression of any of the three heterodimers. There was an inverse association between the presence and levels of HER2 heterodimers and hormone receptor expression in HER2+ tumours. Tumours exhibiting high levels of HER2 heterodimers demonstrated aggressive clinicopathological features and poor outcome. In the HER2+ cases, dimerisation with EGFR and HER3 but not with HER4 showed an association with aggressive features. There was no association between HER2 heterodimers with patient breast cancer-specific survival or recurrence in HER2+ breast cancer in those patients receiving trastuzumab or not. Our results demonstrate that HER2 dimerisation is a complex process that may underlie the biological heterogeneity of HER2 positive tumours and may identify patients suitable for a specific targeted therapy but does not predict patient outcome for those receiving trastuzumab. PLA proved to be a useful tool for detecting, visualising and quantifying the frequency of protein–protein interactions in archival formalin-fixed paraffin-embedded tissue samples.

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Abbreviations

HER:

Human epidermal growth factor receptor

RTKs:

Receptor tyrosine kinases

EGF:

Epidermal growth factor

STATs:

Signal transducer and activation of transcription

PI3K:

Phosphatidylinositol (PtdIns) 3-kinase

IHC:

Immunohistochemistry

ER:

Oestrogen receptor

NPI:

Nottingham Prognostic Index

PgR:

Progesterone receptor

TN:

Triple negative phenotype

ASCO:

American Society of Clinical Oncology

CISH:

Chromogenic in situ hybridisation

TMA:

Tissue microarray

PLA:

In situ proximity ligation assay

HRP:

Horse radish peroxidase

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Acknowledgments

We would like to thank the University of Nottingham for funding the Studentship.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Molecular Pathology Research Unit, Division of Oncology, School of Medicine, Nottingham City Hospital, University of Nottingham, Nottingham, NG5 1PB, UK

    Fabrício F. T. Barros, Christopher C. Nolan, Alice C. Durham, Emad A. Rakha, Ian O. Ellis & Andrew R. Green

  2. Department of Clinical Oncology, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, NG5 1PB, UK

    Tarek M. A. Abdel-Fatah, Paul Moseley & Stephen Chan

  3. Department of Histopathology, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, NG5 1PB, UK

    Emad A. Rakha & Ian O. Ellis

Authors
  1. Fabrício F. T. Barros
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  4. Christopher C. Nolan
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Correspondence to Andrew R. Green.

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Barros, F.F.T., Abdel-Fatah, T.M.A., Moseley, P. et al. Characterisation of HER heterodimers in breast cancer using in situ proximity ligation assay. Breast Cancer Res Treat 144, 273–285 (2014). https://doi.org/10.1007/s10549-014-2871-4

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  • DOI: https://doi.org/10.1007/s10549-014-2871-4

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