Human epidermal growth factor receptor 2 (HER2) plays an important role in breast cancer progression and provides predictive information for response to targeted therapy including trastuzumab although this is limited. Downstream pathways, such as PI3K/Akt, are associated with HER2/HER3 heterodimerization promoting survival and proliferation amongst cancer cells. Thus, patient outcome and trastuzumab therapy effectiveness might be further characterised by HER2/HER3 dimerisation and its signalling pathways. HER2/HER3 dimerisation status was assessed, using chromogenic in situ Proximity Ligation Assay, in two breast cancer series: early stage primary breast cancer, including 224 HER2+ patients that were not submitted to trastuzumab, and HER2+ breast cancer where patients were treated with adjuvant trastuzumab (n = 143). Levels of biomarkers including PI3K, pAKT, ER, PgR, HER3, BCL2, p53, PTEN and p21 were measured using immunohistochemistry. Levels of HER2/HER3 heterodimers were compared with biomarker expression and patient outcome. An association between high levels of HER2/HER3 dimerisation and absence of hormone receptors, ER and PgR, was observed. We further show for the first time the presence of HER2/HER3 heterodimers and the loss of p21 expression in HER2+ breast cancer predicts a significantly poorer outcome when submitted to adjuvant trastuzumab. Breast cancer patients that reveal high levels of HER2/HER3 dimerisation and loss of p21 are associated with poor survival prognosis in patients with HER2+ breast cancer treated with adjuvant trastuzumab. Further quantification analysis of HER dimer/ligand complexes and downstream signalling pathways will begin to unravel the complex associations with patient outcome and its relationship with sensitivity to targeted treatment.
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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