Targeting within ER positive early breast cancer : patient selection for current therapies and novel therapeutic approaches in a heterogeneous group

Over 1 million women a year are diagnosed with Breast Cancer. The majority, approximately 70% express the oestrogen receptor (ER). ER positive breast cancer has historically been perceived as a ‘good cancer’, although many woman with ER+ breast cancer still succumb to their disease and globally breast cancer is the leading cause of female cancer deaths. The advent of gene expression profiling and the definition of the molecular instrinsic subtypes has defined at least two subtypes of ER positive breast cancers (luminal A and luminal B) that differ markedly in terms of biological behaviour, response to adjuvant therapies and most importantly patient outcome. The focus of this research is ER+ breast cancer and targeting patient therapy in this heterogeneous group. This work attempts to translate our understanding of the biology of the ER and cell signalling interactions to aid the correct identification of patients for both current therapy and more novel therapeutic approaches. Following a hypothesis generating pilot study examining whether the level of ER influences response to endocrine therapy, 557 formalin fixed paraffin embedded (FFPE) breast cancer specimens retrieved at time of definitive surgery from early breast cancer patients with available accurate 15 years follow up data were analysed to measure ER, Progesterone receptor (PgR), HER2 and Ki67 expression using immunohistochemistry. Tumour expression of ER, PgR and the combined endocrine receptor (CER), which considers the expression level of both hormone receptors and hypothesised to more accurately quantify endocrine responsiveness by acting as a surrogate marker of a functioning ER signalling pathway, were analysed. The results suggest that in this cohort of ER+ endocrine treated patients CER is a better predictor of endocrine response than either the ER of PgR independently. The CER was thereafter utilised as a surrogate marker of oestrogen receptor signalling pathway to develop a scoring system which included HER2 IHC expression and tumour histological grade, as surrogate markers of the 3 key pathways (ER signalling, HER2 signalling and proliferation). These were chosen as previous studies comparing various gene prognostic profiles indicate commonality in sampling groups of the genes representing their activation. The scoring system, named the Clinical Outcome Score (COS) was developed to represent a pragmatic equivalent of gene prognostic profiles utilising currently routinely measured tumour markers. We hypothesised that COS as an indicator of tumour biology may aid identification of risk in the very challenging group, ER+/HER2 negative patients with intermediate grade and low disease burden, and may help guide adjuvant therapy decisions particularly the indication for chemotherapy . In this exploratory analysis, the distribution of COS scores (2-10) followed a linear response with a notable separation between low scores (2-4) and high scores (5-10). Importantly, when analysed in combination with tumour burden, low COS may help identify patients with nearly 100% long term survival, however in all analysis high COS was associated with a highly significant poorer outcome in terms of early recurrence, late recurrence and 15 year breast cancer specific survival. This group of high risk ER+ breast cancer patients represent a real challenge (and concern) in the treatment of early breast cancer, as there is increasing evidence that ER+ tumours are relatively chemo-in senstive and the response to chemotherapy agents is limited. As a secondary analysis, within our cohort of ER+/ HER2- endocrine treated patients we retrospectively analysed the benefit of chemotherapy in patients with low and high COS scores and the results indicate lack of benefit in the cohort of patients diagnosed 1995-1998. Investigating novel therapeutic targets focusing on the subtypes of breast cancer, and tumour biology involved in endocrine resistance is now beginning to take precedence in breast cancer research. Two potential new therapeutic targets in ER+ breast cancer were studied. The first is the sodium iodide symporter, NIS, a transmembrane glycoprotein which has been exploited for the safe delivery of radio-iodide in the treatment of thyroid cancers for many years. NIS is expressed in many breast cancers, however most breast cancers expressing NIS lack functional uptake as demonstrated by scintography studies and in vivo animal work. In vitro results suggest that the ER is important in NIS regulation and function. In addition MAPK and PI3K-Akt signalling pathways may have a role in NIS regulation- both these pathways are often activated in ER+ breast cancer and known to have extensive crosstalk with the ER. Utilising ER+ and ER negative breast cancer cell lines we examined NIS function following gene delivery with a human NIS (hNIS) transfected plasmid and assessed function and expression of NIS following ER knockdown by siRNA. Our results suggest that the ER phenotype is important but not necessarily the ER per say. We examined NIS expression in a mixed ER+ and ER- cohort (n=50) of patient tumour samples using real time RT-PCR, and report high levels of NIS mRNA expression was limited to ER+ breast tumours. Prompting analysis of NIS expression, cellular location and correlations with cell signalling proteins in 300 ER+ breast cancers using IHC . Significant correlations were identified with key members of the PI3K-Akt and MAPK supporting their role in NIS regulation in vivo. Importantly, in both patient cohorts NIS was found to be significantly associated with poor outcome, and we hypothesis that this is an effect of enhanced growth factor signalling and activation of pathways in biologically more aggressive ER+ cancer (ER+/PgR-) may also regulate NIS and suggest future directions of research. Lastly, as a pilot study expression of Src kinase, a non receptor tyrosine kinase implicated in tamoxifen resistance and breast cancer virulence, was analysed by IHC in the ER+ breast cancer patient cohort. Interestingly nuclear Src kinase was found to be associated with improved outcome and hypothesise that Src Kinase expression in breast cancer may have varying roles in the different subtypes of breast cancer, an important consideration as Src Kinase inhibitors are currently in clinical trials. This pilot study formed a hypothesis that was subsequently examined in another student’s PhD thesis.