Abstract PR-6: Pharmacodynamic analysis of ENCORE 301, a placebo-controlled, randomized phase 2 study of exemestane with and without entinostat in postmenopausal ER+ breast cancer patients demonstrates an association of lysine hyperacetylation with clinical outcome.
Authors: Ordentlich Peter 
Published: June 2012

Abstract
Despite promising preclinical data and extensive clinical testing, histone deacetylase inhibitors (HDACi) as a class have not demonstrated significant activity in solid tumors as single agents or in combination. Even where HDACi have proven to be effective e.g. T-cell lymphomas, there is still an inability to identify those patients most likely to benefit due to a lack of association between outcome and acetylation. We report that pharmacodynamic (PD) analysis of patient samples from ENCORE-301, a recently completed randomized phase 2 placebo controlled study of exemestane with and without the HDACi entinostat in post-menopausal breast cancer patients (n=130), demonstrates an association of HDACi-induced lysine hyperacetylation with improved clinical outcome.
Protein lysine acetylation was measured in circulating B cells (B), T cells (T) and monocytes (M) by multi-parameter flow cytometry from samples taken at pre-treatment, D1, D8, and D15 of cycle 1 from patients treated with exemestane plus entinostat (EE) or exemestane plus placebo (EP). Percent change was calculated and related to progression free survival (PFS) outcome data. Hyperacetylation independent of treatment arm is defined as a percent change increase above the calculated median percent change for each cell type.
Pre- and post treatment samples were obtained in a subset of 49 patients (EE = 27; EP = 22). Review of baseline characteristics in this subset indicates that they appear to be consistent with the entire study population. Hyperacetylation across all cell types in EE vs EP was associated with prolonged median PFS (B: 8.54 months vs 1.92 HR=0.24 (95% CI 0.081, 0.690); T: 6.57 vs 1.77 HR=0.24 (95% CI 0.087, 0.640); M: 6.21 vs 1.87 HR=0.50 (95% CI 0.211, 1.203). Preliminary trends in overall survival also favor the EE hyperacetylation group. Samples taken for plasma concentration measurements of entinostat indicate that entinostat levels at the D8 and D15 timepoints used for the PD analysis were generally at or below the assay detection limits (&lt; 0.5 ng/ml) preventing a correlation to be made between acetylation increase and entinostat concentration. Characterization of adverse events with 10% or greater difference between treatments in the ENCORE-301 safety population (n = 129) in the 49 patient biomarker patient subset indicates that thrombocytopenia incidence may be associated with hyperacetylation in the EE group while incidence of other AEs including fatigue do not appear to be associated with hyperacetylation.
These data provide for the first time a clear association of HDACi induced protein lysine hyperacetylation and clinical outcome. Several factors may contribute to the success in demonstrating this association including the randomized, controlled study design, positive outcome of ENCORE-301 and a sensitive pharmacodynamic assay that allows for measurement of global protein lysine acetylation changes. Combined with the overall positive results of ENCORE-301 (median PFS EE vs EP 4.28 vs 2.27 months HR 0.73 (95% CI 0.49, 1.09); and OS with median follow up of 18 months EE vs EP 26.9 vs 20.3 months HR 0.56 (95% CI 0.31, 1.02)), these data provide evidence of a potential breakthrough in the expansion of epigenetic therapy to solid tumors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-6.