The use of proximity ligation assay to identify HER2:HER3 heterodimers in early breast cancers.

Abstract

Abstract Abstract #2068 Background: The HER receptor family includes four members: HER1, HER2, HER3 and HER4. Expression of these proteins is linked to breast cancer development and progression. HER2 gene amplification is found in 20-30% of breast cancers. Overexpression of HER2 has been linked with high grade tumours and poor prognosis. Clinical studies have demonstrated a strong relationship between HER2 and HER3 with 70% of HER3-positive tumours also being positive for HER2. Co-expression of HER2/HER3 has been suggested to drive endocrine and chemotherapy resistance. However such resistance is driven not by co-expression but by signalling via HER2/HER3 dimers which have previously been impossible to detect in situ. Proximity ligation assays (PLAs) can detect homo- and heterodimers in situ at a single-molecule level and allow study of signalling pathways. Paired antibodies are used to detect protein complexes in tissues.
 Methods: Using tissue microarrays containing 100 breast cancer samples, we analysed HER2 homodimers and HER2:HER3 heterodimers by PLA. HER2 amplification was detected using FISH analysis.
 Results: PLA was used to detect HER2 homo and heterodimers in vivo and in vitro. HER2 homodimers were detected in amplified and non-amplified cases. A significant association (p<0.00001) was identified between HER2 homodimerisation and amplification. HER2 homodimerisation was not associated with any clinicopathological data.
 HER2:HER3 heterodimers were detected in vivo and in vitro through PLA and western blot analysis. The median number of HER2:HER3 heterodimers (7.8 signals per cell) was greater than HER2:HER2 homodimers (2.0 signals per cell) in this series indicating HER2:HER3 may be the preferred dimer. HER2:HER3 was significantly associated with nodal status (p=0.003) and grade (p=0.02).
 Conclusions: Using an in situ PLA we demonstrated the in vivo expression of HER2 homodimers in human breast cancer and increased HER2 homodimerisation linked to HER2 gene amplification. We have demonstrated HER2:HER3 heterodimers to be linked to nodal status and grade. Our observations suggest that PLA is an invaluable method to detect homo and heterodimers in situ and could potentially be used a diagnostic tool in the future. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2068.