Characterisation of HER heterodimers in breast cancer using in situ proximity ligation assay.

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.

HER2/HER3 heterodimers and p21 expression are capable of predicting adjuvant trastuzumab response in HER2+ breast cancer.

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.

Calpain-1 expression is associated with relapse-free survival in breast cancer patients treated with trastuzumab following adjuvant chemotherapy.

The calpain family, and their endogenous inhibitor calpastatin, has been implicated in cancer progression, and recent in vitro data have indicated a role in trastuzumab resistance. The aims of our study were to examine expression levels of calpastatin, calpain-1 and calpain-2 in breast tumours from patients treated with trastuzumab following adjuvant chemotherapy to determine their potential as biomarkers to predict therapeutic response. The expression of calpastatin, calpain-1 and calpain-2 was determined, using immunohistochemistry (IHC), in tumours from a series of 93 patients with primary breast cancer treated with surgery and adjuvant chemotherapy with or without trastuzumab followed by trastuzumab to complete 1 year of therapy. IHC was performed using tissue microarrays constructed from cores taken from intratumour and peripheral tumour areas. Expression was correlated with clinicopathologic variables and patient outcome. Calpastatin expression was correlated with Nottingham prognostic index (p = 0.003) and lymph node status (p = 0.007). Trastuzumab resistance was defined as disease relapse during therapy. Calpain-1 expression is associated with relapse-free survival (p = 0.001) and remained significant in multivariate analysis accounting for confounding pathological and treatment variables (hazard ratio 4.60, 95% confidence interval 1.05-20.25; p = 0.043). Calpain-1 may be a useful biomarker to predict relapse-free survival in breast cancer patients treated with adjuvant trastuzumab and chemotherapy. A larger verification study is warranted.

Understanding the HER family in breast cancer: interaction with ligands, dimerization and treatments.

Breast carcinoma is the most frequent type of cancer affecting women. Among the recently described molecular and phenotypic classes of breast cancer, human epidermal growth factor receptor 2 (HER2)-positive tumours are associated with a poor prognosis. HER2 plays an important role in cancer progression being targeted to provide predictive and prognostic information. Moreover, HER2 is related to cancer resistance against a variety of therapies; however, trastuzumab (herceptin) has proved successful in treatment of this subgroup. Nevertheless, resistance to this drug may be acquired by patients after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerization between members of the HER family may contribute to resistance against treatments due to different combinations that trigger different downstream pathways. This is promoted by ligands, which are expressed as transmembrane precursor protein molecules and have a conserved epidermal growth factor-like domain. Through resistance to trastuzumab, other drugs are being developed to interact in different domains of HER2 protein. It might be a good strategy to apply new drugs simultaneously to trastuzumab due to act in different domains of HER2. The study of interaction between receptors/ligands will characterize specifically their signalling pathway and understand which strategy to acquire.