Barros, Fabrício Félix Tabuada
(2013)
Biological characterisation of HER2 amplified breast cancer.
PhD thesis, University of Nottingham.
Abstract
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 status is currently assessed in routine breast cancer reporting using immunohistochemistry (IHC) in addition to in situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to predict response to targeted therapy (Trastuzumab) is well documented. However, prognostic information provided by IHC expression categories and prognostic value added by using ISH in borderline cases remains unclear. 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 has proved successful in treatment of this subgroup. Nevertheless, patients may acquire resistance to this drug after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerisation 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. The study of interaction between receptors/ligands will characterise specifically their signalling pathway and understand which strategy to acquire.
The main aim of this thesis was to assesses the status of HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER heterodimers (in situ proximity ligation assay (PLA)), including HER2/EGFR, HER2/HER3 and HER2/HER4, in two BC series prepared in tissue microarray format; a series of consecutive primary operable BC cases (n = 1858) including HER2+ trastuzumab naïve cases (TrN, n = 221), the second series of HER2+ trastuzumab adjuvant treated cases (TrT, n = 143). Therefore determining the biological characterisation of these biomarkers by associating against clinicopathological parameters, survival outcome and understand the trastuzumab therapy value.
There was excellent overall concordance between HercepTest negative (scores 0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17 copy number ratio of ≥ 2; p < 0.001). Kaplan-Meier analysis for breast cancer specific survival (BCSS) and disease free interval (DFI) revealed statistically significant differences between HER2 positive and negative cases detected by HercepTest and CISH (p < 0.001). Interestingly, it was identified that HercepTest 2+ non-amplified cases were not significantly different with those amplified 2+ or 3+ cases with respect to their behaviour (BCSS and DFI).
The results revealed an inverse association between the HER heterodimerisation status and hormone receptor status (p < 0.001), and a significantly worse outcome amongst cases revealing high levels of all heterodimers (p < 0.001). Among ER+ cases, the heterodimers high levels were significantly associated with worse prognosis (p < 0.001) overall. However amongst the two HER2+ populations dimerisation status did not show an association with patient outcome.
The overall concordance between HercepTest and CISH analysis for HER2 status was excellent. All HercepTest 2+ cases identified were observed to have poor outcomes similar to those HercepTest 3+ cases regardless of gene amplification status. In the current clinical environment cases exhibiting IHC 2+ and non-amplified gene HER2 status will not be offered targeted HER2 therapy but do exhibit aggressive clinical behavioural characteristics. Even though those patients with high levels of the HER2 truncated form, p95HER2, have shown poor outcome, this biomarker does not reveal any extra findings comparing with the HER2 expression results. Beside HER2, EGFR is the only monomer that reveals prognostic value amongst the breast cancer patients. Tumours exhibiting high levels of HER heterodimerisation have an adverse prognosis, however in the context of HER2+ breast cancer no association with clinical outcome was observed regardless of use of trastuzumab treatment. HER2/HER3 heterodimerisation status assessed in multivariate analyses has shown that this protein-protein interaction is associated with a poor prognostic outcome, which needs further investigation and assessment of clinical utility to use in the future in breast cancer treatment decision. Further quantification analysis of dimer/ligand complex using PLA of other HER family members may be useful to identify subset of patients associated with distinct outcome, response to treatment and relationships with HER signalling related biomarkers.
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