Breast Cancer Research and Treatment

, Volume 132, Issue 2, pp 463–470 | Cite as

In situ detection of HER2:HER2 and HER2:HER3 protein–protein interactions demonstrates prognostic significance in early breast cancer

  • Melanie Spears
  • Karen J. Taylor
  • Alison F. Munro
  • Carrie A. Cunningham
  • Elizabeth A. Mallon
  • Chris J. Twelves
  • David A. Cameron
  • Jeremy Thomas
  • John M. S. BartlettEmail author
Preclinical Study


HER2 overexpression/amplification is linked with poor prognosis in early breast cancer. Co-expression of HER2 and HER3 is associated with endocrine and chemotherapy resistance, driven not simply by expression but by signalling via HER2:HER3 or HER2:HER2 dimers. Proximity ligation assays (PLAs) detect protein–protein complexes at a single-molecule level and allow study of signalling pathways in situ. A cohort of 100 tumours was analyzed by PLA, IHC and FISH. HER complexes were analyzed by PLA in a further 321 tumours from the BR9601 trial comparing cyclophosphamide, methotrexate and fluorouracil (CMF) with epirubicin followed by CMF (epi-CMF). The relationships between HER dimer expression and RFS and OS were investigated, and multivariate regression analysis identified factors influencing patient prognosis. PLA successfully and reproducibly detected HER2:HER2 and HER2:HER3 protein complexes in vivo. A significant association (P < 0.00001) was identified between HER2 homodimerization and HER2 gene amplification. Following a minimum p value approach high levels of HER2:HER2 dimers were significantly associated with reduced relapse-free (RFS; hazard ratio = 1.72, 95% confidence interval 1.15–2.56, P = 0.008) and overall survival (OS HR = 1.69 95% CI = 1.09–2.62, P = 0.019). Similarly, high levels of HER2:HER3 dimers were associated with reduced RFS (HR = 2.18, 95% CI = 1.46–3.26, P = 0.00016) and OS (HR = 2.21, 95% CI = 1.41–3.47, P = 0.001). This study demonstrates that in situ detection of HER2 and HER2:3 protein:protein complexes can be performed robustly and reproducibly in clinical specimens, provides novel prognostic information and opens a significant novel opportunity to probe the clinical impact of cellular signalling processes.


Breast cancer HER2 HER3 Proximity ligation assay 


Conflict of interest

All authors state that they have no financial conflicts of interest.


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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Melanie Spears
    • 1
    • 2
  • Karen J. Taylor
    • 1
    • 2
  • Alison F. Munro
    • 2
  • Carrie A. Cunningham
    • 2
  • Elizabeth A. Mallon
    • 3
  • Chris J. Twelves
    • 4
  • David A. Cameron
    • 5
  • Jeremy Thomas
    • 1
  • John M. S. Bartlett
    • 1
    • 6
    Email author
  1. 1.Edinburgh Breakthrough Breast Cancer Unit, Edinburgh Cancer Research CentreWestern General HospitalEdinburghUK
  2. 2.Endocrine Cancer Research Group, Edinburgh Cancer Research CentreWestern General HospitalEdinburghUK
  3. 3.Department of PathologyWestern InfirmaryGlasgowUK
  4. 4.Leeds Institute of Molecular Medicine and St James’s Institute of OncologyLeeds Cancer Research UK CentreLeedsUK
  5. 5.Edinburgh Cancer Research CentreWestern General HospitalEdinburghUK
  6. 6.Breakthrough Breast Cancer, Edinburgh Cancer Research CentreWestern General HospitalEdinburghUK

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