Sequencing analysis reveals evidence of immune activation in advanced HER2 negative breast cancer responders treated with entinostat + nivolumab + ipilimumab

We analyzed the pathways associated with therapeutic response in 19 patients (5 responders) enrolled in a Phase Ib trial (NCT02453620) diagnosed with advanced HER2-negative breast cancer and treated with the HDAC inhibitor entinostat, in combination with dual immune checkpoint inhibitors (ICIs), nivolumab and ipilimumab. The analysis included gene expression and T-cell receptor (TCR) repertoire data derived from bulk RNA-seq and neoantigen data from whole exome sequencing. A total of 37 tumor biopsies were taken from six different metastatic sites at three timepoints: before treatment (baseline), after a two-week entinostat run-in (C1D1), and after the dual ICIs were added (week 8). Paired differential gene expression (DE) analysis between different timepoints revealed changes in immune-related pathways, such as interferon gamma and inflammatory response, IL6/JAK/STAT3 and IL2/STAT5 signaling, and allograft rejection after week 8 of triplet treatment. The DE analysis of response at each timepoint also revealed significant changes at baseline in immune-related pathways, such as inflammatory and interferon alpha and gamma responses, suggesting that responders may have had a preexisting immune tumor microenvironment (TME) that primed them for triplet therapy. Further analysis of the TME revealed significantly higher expression of genes representative of subsets of CD8+ T and plasma cells after entinostat treatment, indicating potential mechanisms of TME sensitization by HDACi. Genes representative of M1-like macrophages, pDC, memory CD4+ T, B, NK, lymphatic endothelial, Th1 cells, and memory B cells were significantly altered in responders after triplet treatment. PAM50 molecular subtype analysis revealed that basal and luminal B subtypes correlate with response. TCR analysis demonstrated higher diversity, and exploratory analysis of strong binding neoantigens showed fewer strong neoantigen binders in responders at week 8. In summary, the sequencing analysis reveals important changes in the immune landscape of responders to the combined treatment of entinostat and ICIs and suggests that pretreatment with entinostat may sensitize the immune TME to promote response.