Abstract P2-13-39: Investigation of neratinib and endocrine therapy combinations in HER2 positive breast cancer models
Authors: Mahdi Amira F , Conlon Neil T , Eli Lisa D , Diala Irmina , Crown John , Collins Denis 
Published: March 2022

Abstract
Background: HER2-positive (+) breast cancer (BC) represents a significant clinical burden due to an aggressive phenotype and propensity for brain metastasis. Up to 70 % of HER2+ BCs are also oestrogen receptor (ER)-positive, with receptor crosstalk often attributed to anti-HER2 therapeutic resistance. Neratinib is a pan-HER tyrosine kinase inhibitor, currently approved for the treatment of HER2+ BC. Dual-blockade with anti-HER2 and endocrine therapies (ETs) has shown positive outcomes in clinical trials, without the added toll of traditional chemotherapy. Fulvestrant is the only currently approved selective ER degrader (SERD), but it is hindered by poor bioavailability, leading to several orally available SERDs being developed. CDK4/6 inhibitors have been approved for ER+ BC, and trials are ongoing to see if their addition also benefits ER+/HER2+ BC patients. The aim of this study is to outline the benefit of ETs in HER2+ BC and to investigate neratinib/ET combinations with potential for the treatment of HER2+/ER+ BC. Methods: BC patient overall (OS) and recurrence free (RFS) survival and clinical data for a number of independent breast cancer studies was sourced from the METABRIC study via cBioportal and from KMplotter. Patients were stratified according to HER2 status, ER status and treatment with ET. GraphPad Prism 8 was then used to produce Kaplan-Meier graphs and to calculate difference in patient survival according to treatment with ET. Fulvestrant (SERD, #S1191), AZD9496 (novel, orally bioavailable SERD, #S8372) and ribociclib (CDK4/6 inhibitor, #S7440) were obtained from Selleckchem. Neratinib was obtained from Puma Biotechnology, Inc. Estradiol (Sigma, #E2758) treatment was carried out at 1nM. To assess drug effect, the HER2+/ER+ BC cell line BT-474 was treated for 5 days before growth inhibition was measured using an acid phosphatase-based proliferation assay. Compounds were combined at fixed ratios. Drug IC50 and combination index (CI) values were calculated using Calcusyn. Results: Analysis of the METABRIC study shows HER2+ BC patients who receive ET, numerically experience a longer median survival, with particular separation seen in early OS (ET = 113.8 months vs no ET= 85.50 months, HR= 0.8797, 95% CI: 0.6402 - 1.209, p=0.0987). In addition, analysis of KMPlotter data shows HER2+ patients who receive ET may also experience greater 5 year RFS (HR= 0.7639, 95% CI: 0.5452 to 1.070, p= 0.0539). Despite this potential benefit, the METABRIC dataset showed within a cohort of HER2+ patients, 10.5% were ER+ but did not receive ET. An in vitro cell line model of HER2+/ER+ BC was highly sensitive to neratinib (N) at nanomolar concentrations (IC50 = 2.0549± 0.7 nM). In contrast, fulvestrant (F), AZD9496 (A) and ribociclib (R) were less effective as single agents and an IC50 was not achieved (% growth @ 5µM: F = 54.7 ± 8.5%, A = 61.3 ± 7.8% and R = 95.6 ± 8.7%). The combination of N with F was compared to N with A. Both combinations demonstrated synergy, defined as CI &lt; 1 @ ED50 (N+F CI value = 0.21385 ± 0.15, N+A CI value = 0.25448 ± 0.26). The combination of N + R did not improve the efficacy of N alone ( % growth at max, N = 6.8 ± 1.7% vs N +R = 10.8 ± 2.8%, p&gt;0.9999). Further, triplet combinations with R did not significantly affect cell growth for either the N + F or the N + A combinations (% growth @ max conc., N + F= 5.8 ± 1.6%, N+F+R = 7.2 ± 1.2 %, N+A= 8.2 ± 2%, N+A+R= 9.8 ± 1.7%, p&gt;0.9999). Estradiol supplementation at time of drug treatment significantly increased the growth inhibitory effect of A (p = 0.0489 @ 5µM). Conclusions: Using publicly available data, our results highlight the positive impact ET has on HER2+ breast cancer survival outcome. In vitro data supports the combination of novel SERDs and neratinib as a therapeutic strategy that warrants further investigation in HER2+/ER+ breast cancer.
Citation Format: Amira F Mahdi, Neil T Conlon, Lisa D Eli, Irmina Diala, John Crown, Denis Collins. Investigation of neratinib and endocrine therapy combinations in HER2 positive breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-39.