title: In situ detection of HER2:HER2 and HER2:HER3 protein-protein interactions demonstrates prognostic significance in early breast cancer.
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.
authors.affiliation: Edinburgh Breakthrough Breast Cancer Unit, Edinburgh Cancer Research Centre, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, UK.
authors.forename: Melanie
authors.initials: M
authors.lastname: Spears
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Taylor
authors.forename: Alison F
authors.initials: AF
authors.lastname: Munro
authors.forename: Carrie A
authors.initials: CA
authors.lastname: Cunningham
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Mallon
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Twelves
authors.forename: David A
authors.initials: DA
authors.lastname: Cameron
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thomas
authors.forename: John M S
authors.initials: JM
authors.lastname: Bartlett
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000235:genetics / Q000401:mortality / Q000473:pathology; D018572:Disease-Free Survival; D005260:Female; D005784:Gene Amplification; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D053208:Kaplan-Meier Estimate; D008875:Middle Aged; D015999:Multivariate Analysis; D009367:Neoplasm Staging; D016016:Proportional Hazards Models; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D016032:Randomized Controlled Trials as Topic; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000235:genetics; D020893:Receptor, ErbB-3 / Q000032:analysis* / Q000235:genetics; D018570:Risk Assessment; D012307:Risk Factors; D013997:Time Factors; D016896:Treatment Outcome
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; C508053:ERBB2 protein, human