Abstract P2-09-37: Immunohistochemical Markers Progesterone Receptor, HER2, Ki67 and bcl-2-Associated Athanogene 1 and Prediction of Adjuvant Tamoxifen Treatment Outcome in ER+ Early Breast Cancer
Authors: Gee JM , Aleskandarany MA , Finlay P , Farrow L , Nicholson RI , Habashy HO , Green AR , Rakha EA , Powe DG , Jasani B , Barrett-Lee PJ , Robertson JF , Shaw VE , Ellis IO. 
Published: April 2011

Background: Gene signatures including OncotypeDX are under intense study for their prognostic value, but there is also interest in their predictive value for outcome on endocrine therapy and whether markers reflected by such signatures retain predictive value when measured immunohistochemically (IHC). OncotypeDX comprises 16 cancer (& 5 reference) genes, including oestrogen receptor (ER)-related genes, HER2 pathway and proliferation/cell cycle-associated genes. A further gene in OncotypeDX, bcl-2-associated athanogene 1 (BAG-1), is understudied in breast cancer but increased IHC for BAG-1 has recently been related to improved tamoxifen outcome (Millar et al. Br J Cancer 100, 123-33, 2009). Here we examine if IHC for progesterone receptor (PR), HER2 and the proliferation-associated protein Ki67 can discriminate adjuvant tamoxifen outcome in an ER+ primary breast cancer series. We also evaluate BAG-1 in this series, and in ER+ breast cancer cell lines during antihormone treatment and following acquisition of resistance, to further consolidate relation of BAG-1 IHC to antihormone outcome. Material and Methods: IHC for PR, HER2, Ki67 (DAKO clone MIB1) and BAG-1 (Chemicon MAB4611) was performed in FFPE tissue microarrays from tamoxifen-treated ER+ primary breast cancer patients with 10 year follow-up (n=383). Clinicopathological data, including disease-free interval (DFI) and breast cancer-specific survival (BCSS), were available for statistical analysis. BAG-1 IHC was also performed on paraffin-embedded cell pellets prepared from ER+ breast cancer lines (MCF-7, T47D) after 10 days culture with 10-7M 4OH-tamoxifen (4OHT) and following acquisition of resistance.

Results: Kaplan Meier analysis demonstrated that increased NPI, PR negativity, HER2 overexpression and Ki67 positivity (&gt;=10%) could discriminate ER+ tamoxifen-treated patients with reduced DFI (P&lt;0.001, p=0.013, 0.004, p=0.002 respectively) and shortened BCSS (P&lt;0.001, P&lt;0.001, p=0.004, P&lt;0.001 respectively). Patients with the highest Ki67 (&gt;70%) had significantly poorer outcome (DFI & BCSS: P&lt;0.001). Multivariate analysis confirmed these IHC markers were independent predictive factors for DFI and BCSS in this tamoxifen-treated series. Kaplan Meier analysis showed tamoxifen-treated patients with reduced/no nuclear BAG-1 (&lt;30% cut-off) had decreased DFI (p=0.046) and BCSS (p=0.018). Tumours with lower BAG-1 also had reduced levels of ER (p=0.033), PR (P&lt;0.001) and of luminal cytokeratins 7/8 (p=0.049) and 19 (p=0.022), with increased tumour grade (p=0.043). Patients whose tumours were Ki67 positive with reduced/no BAG-1 also had a poorer outcome than their BAG-1 high counterparts (DFI p=0.073; BCSS p=0.034). In the ER+ models, BAG-1 immunostaining was increased by initial 4OHT treatment, but barely detectable in their acquired resistant sublines. Discussion: Along with PR and HER2, IHC for Ki67 and BAG-1 may be useful in predicting outcome of adjuvant tamoxifen-treated patients and should be examined in future validation studies. BAG-1 loss also hallmarks, and could contribute to mechanisms of, acquired tamoxifen resistance in breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-37.