Abstract P4-15-01: Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors
Authors: Leal MF , Haynes BP , Schuster E , Yeo B , Afentakis M , Dodson A , Buus R , Cheang MC , Martin L-A , Dowsett M 
Published: May 2019

Abstract
Background: Aromatase inhibitors (AI), which block the conversion of androgen to estrogen, are the mainstay therapy for estrogen receptor–positive (ER+) breast cancer (BC). However, many patients relapse.
Purpose: To identify molecular alterations associated with long-term neoadjuvant AI therapy of ER+ BC.
Methods: We retrospectively identified 137 patients diagnosed with ER+ BC and treated with neoadjuvant AI for at least 1 month at the Royal Marsden Hospital (2003–2016). Paired pre- (diagnosis) and post-AI (surgery) biopsies with &gt;40% invasive cell areas were available from 87 ER+ BC patients. In all samples, we evaluated ER, PR and Ki67 immunostaining, ESR1 hot-spot mutations by droplet digital PCR and expression of 801 genes associated with BC and response to endocrine therapy by NanoString.
Results: Mean time on neoadjuvant treatment was 26 weeks (range 5.6–92.3). Cell proliferation remained suppressed in most tumours indicating little evidence for acquired resistance: 56/87 (64%) showed residual Ki67 (Ki67r) &lt;2.7% (complete cell cycle arrest) and 14/87 (11.5%) had Ki67r&gt;10%. This was paralleled by reduced expression of proliferation genes, ER (immunostaining and gene expression) and estrogen-regulated genes (ERG) at surgery compared with the diagnostic samples (all p&lt;0.001). There was a weak positive correlation between AI duration and Ki67r and less reduction in proliferation genes, ESR1 and ERGs expression (p&lt;0.05; r=0.26-0.34). Pathway analysis revealed inhibition of cell cycle (e.g. reduced expression of several cyclins), E2F targets and estrogen response (e.g. reduced expression of ER downstream targets such as FOXM1). CDK genes showed a variable response: CDK1 and CDK2 decreasing, CDK4 increasing and no change in CDK6. Most notably, 6 surgical samples showed ESR1 mutations: one of these cases had the mutation at diagnosis. All 5 acquired mutations were detected in patients treated for &gt;6 months, giving a prevalence in this cohort of 5/34 (15%). Tumours with ESR1 mutations showed less suppression of ERGs (p=0.002) and proliferation (p=0.039) and increased ESR1 (p=0.016) expression at surgery compared with tumours without mutation. Pathway analysis confirmed lack of inhibition of estrogen response (FDR&gt;5%) and less inhibition of cell cycle [enrichment score: -0.49 vs -0.78) and E2F targets (-0.47 vs -0.8) in tumours with mutation. Tumours without ESR1 mutation but with Ki67r&gt;10% also showed reduced ERGs response (p=0.006) compared to tumours with Ki67r&lt;2.7%. Additionally, these tumours showed relative activation of cell cycle, estrogen response, E2F targets pathways and mTORC1 signalling (FDR&lt;1%).
Conclusion: Overall most tumours showed no evidence for the emergence of resistant disease after neoadjuvant AI therapy even after many months of treatment. However, we detected an enrichment of ESR1 mutations (15% of cases) after long-term treatment as a putative driver of ERG expression and proliferation and thus reduced AI response. Therefore, mutant ER appears to be associated with ligand-independent ERG activity supporting the clinical validity of dual blockade with a selective ER down-regulator combined with a CDK4/6 inhibitor targeting the RB/E2F axis in this scenario.
Citation Format: Leal MF, Haynes BP, Schuster E, Yeo B, Afentakis M, Dodson A, Buus R, Cheang MC, Martin L-A, Dowsett M. Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-15-01.