Advertisement

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

Abstract

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.

Keywords

Breast cancer HER2 HER3 Proximity ligation assay 

Notes

Conflict of interest

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

References

  1. 1.
    Abd El-Rehim DM, Pinder SE, Paish CE, Bell JA, Rampaul RS, Blamey RW, Robertson JF, Nicholson RI, Ellis IO (2004) Expression and co-expression of the members of the epidermal growth factor receptor (EGFR) family in invasive breast carcinoma. Br J Cancer 91:1532–1542PubMedCrossRefGoogle Scholar
  2. 2.
    Albanell J, Baselga J (2001) Unraveling resistance to trastuzumab (Herceptin): insulin-like growth factor-I receptor, a new suspect. J Natl Cancer Inst 93:1830–1832PubMedCrossRefGoogle Scholar
  3. 3.
    Aubele M, Spears M, Ludyga N, Braselmann H, Feuchtinger A, Taylor KJ, Lindner K, Auer G, Stering K, Hofler H, Schmitt M, Bartlett JM (2010) In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein-protein complexes in paraffin sections from breast cancer tissues. Br J Cancer 103:663–667PubMedCrossRefGoogle Scholar
  4. 4.
    Bartlett JM, Going JJ, Mallon EA, Watters AD, Reeves JR, Stanton P, Richmond J, Donald B, Ferrier R, Cooke TG (2001) Evaluating HER2 amplification and overexpression in breast cancer. J Pathol 195:422–428PubMedCrossRefGoogle Scholar
  5. 5.
    Bartlett JM, Munro A, Cameron DA, Thomas J, Prescott R, Twelves CJ (2008) Type 1 receptor tyrosine kinase profiles identify patients with enhanced benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial. J Clin Oncol 26:5027–5035PubMedCrossRefGoogle Scholar
  6. 6.
    Citri A, Skaria KB, Yarden Y (2003) The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp Cell Res 284:54–65PubMedCrossRefGoogle Scholar
  7. 7.
    Desmedt C, Sperinde J, Piette F, Huang W, Jin X, Tan Y, Durbecq V, Larsimont D, Giuliani R, Chappey C, Buyse M, Winslow J, Piccart M, Sotiriou C, Petropoulos C, Bates M (2009) Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH. Diagn Mol Pathol 18:22–29PubMedCrossRefGoogle Scholar
  8. 8.
    Edwards J, Krishna NS, Grigor KM, Bartlett JM (2003) Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer. Br J Cancer 89:552–556PubMedCrossRefGoogle Scholar
  9. 9.
    Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477PubMedCrossRefGoogle Scholar
  10. 10.
    Fredriksson S, Dixon W, Ji H, Koong AC, Mindrinos M, Davis RW (2007) Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nat Methods 4:327–329PubMedGoogle Scholar
  11. 11.
    Hendriks BS, Opresko LK, Wiley HS, Lauffenburger D (2003) Quantitative analysis of HER2-mediated effects on HER2 and epidermal growth factor receptor endocytosis: distribution of homo- and heterodimers depends on relative HER2 levels. J Biol Chem 278:23343–23351PubMedCrossRefGoogle Scholar
  12. 12.
    Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF 3rd, Hynes NE (2003) The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci USA 100:8933–8938PubMedCrossRefGoogle Scholar
  13. 13.
    Kirkegaard T, Witton CJ, McGlynn LM, Tovey SM, Dunne B, Lyon A, Bartlett JM (2005) AKT activation predicts outcome in breast cancer patients treated with tamoxifen. J Pathol 207:139–146PubMedCrossRefGoogle Scholar
  14. 14.
    Kirkegaard T, Edwards J, Tovey S, McGlynn LM, Krishna SN, Mukherjee R, Tam L, Munro AF, Dunne B, Bartlett JM (2006) Observer variation in immunohistochemical analysis of protein expression, time for a change? Histopathology 48:787–794PubMedCrossRefGoogle Scholar
  15. 15.
    Nahta R, Yuan LX, Zhang B, Kobayashi R, Esteva FJ (2005) Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells. Cancer Res 65:11118–11128PubMedCrossRefGoogle Scholar
  16. 16.
    Naresh A, Long W, Vidal GA, Wimley WC, Marrero L, Sartor CI, Tovey S, Cooke TG, Bartlett JM, Jones FE (2006) The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells. Cancer Res 66:6412–6420PubMedCrossRefGoogle Scholar
  17. 17.
    Poole CJ, Earl HM, Hiller L, Dunn JA, Bathers S, Grieve RJ, Spooner DA, Agrawal RK, Fernando IN, Brunt AM, O’Reilly SM, Crawford SM, Rea DW, Simmonds P, Mansi JL, Stanley A, Harvey P, McAdam K, Foster L, Leonard RC, Twelves CJ (2006) Epirubicin and cyclophosphamide, methotrexate, and fluorouracil as adjuvant therapy for early breast cancer. N Engl J Med 355:1851–1862PubMedCrossRefGoogle Scholar
  18. 18.
    Ram TG, Ethier SP (1996) Phosphatidylinositol 3-kinase recruitment by p185erbB-2 and erbB-3 is potently induced by neu differentiation factor/heregulin during mitogenesis and is constitutively elevated in growth factor-independent breast carcinoma cells with c-erbB-2 gene amplification. Cell Growth Differ 7:551–561PubMedGoogle Scholar
  19. 19.
    Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000PubMedCrossRefGoogle Scholar
  20. 20.
    Soderberg O, Leuchowius KJ, Gullberg M, Jarvius M, Weibrecht I, Larsson LG, Landegren U (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45:227–232PubMedCrossRefGoogle Scholar
  21. 21.
    Spears M, Kenicer J, Munro AF, Bartlett JMS (2008) Type I receptor tyrosine kinases as predictive or prognostic markers in early breast cancer. Biomark Med 2:397–407PubMedCrossRefGoogle Scholar
  22. 22.
    Tokunaga E, Kimura Y, Oki E, Ueda N, Futatsugi M, Mashino K, Yamamoto M, Ikebe M, Kakeji Y, Baba H, Maehara Y (2006) Akt is frequently activated in HER2/neu-positive breast cancers and associated with poor prognosis among hormone-treated patients. Int J Cancer 118:284–289PubMedCrossRefGoogle Scholar
  23. 23.
    Tovey SM, Witton CJ, Bartlett JM, Stanton PD, Reeves JR, Cooke TG (2004) Outcome and human epidermal growth factor receptor (HER) 1–4 status in invasive breast carcinomas with proliferation indices evaluated by bromodeoxyuridine labelling. Breast Cancer Res 6:R246–R251PubMedCrossRefGoogle Scholar
  24. 24.
    Tovey S, Dunne B, Witton CJ, Forsyth A, Cooke TG, Bartlett JM (2005) Can molecular markers predict when to implement treatment with aromatase inhibitors in invasive breast cancer? Clin Cancer Res 11:4835–4842PubMedCrossRefGoogle Scholar
  25. 25.
    Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM (2003) Expression of the HER1–4 family of receptor tyrosine kinases in breast cancer. J Pathol 200:290–297PubMedCrossRefGoogle Scholar
  26. 26.
    Worthylake R, Opresko LK, Wiley HS (1999) ErbB-2 amplification inhibits down-regulation and induces constitutive activation of both ErbB-2 and epidermal growth factor receptors. J Biol Chem 274:8865–8874PubMedCrossRefGoogle Scholar

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

Personalised recommendations