Abstract P2-03-01: Akt inhibition associated with change in immunophenotype of tumor microenvironment (TME) in breast cancer (BC)
Authors: Marks DK , Gartrell RD , Pan Q , El Asmar M , Hart TD , Esancy CL , Lu Y , Yu J , Hibshoosh H , Connolly E , Kalinsky K , Saenger YM 
Published: May 2019

Abstract
Background: The PI3K/Akt/mTOR pathway is a known oncogenic pathway in BC. In addition, this pathway has demonstrated capacity to modulate host immune activity and may indirectly affect tumorigenesis. Clinicopathologic studies have demonstrated that lymphocyte density within the TME is predictive of chemosensitivity and improved prognosis in BC, while myeloid infiltration may play a deleterious role. To define the impact of Akt inhibition on the TME, we analyzed tumor tissue from patients (pts) with early-stage BC treated with single agent MK-2206, an Akt inhibitor, enrolled on a presurgical trial (NCT01319539).
Methods: Quantitative immunofluorescence (qmIF) was performed for CD3, CD8, CD4, FOXP3, CD68, Pancytokeratin on 4uM sections from biopsy and surgical specimens of MK-2206 (n=5) and control (n=5) pts. Images were analyzed using Vectra/inForm software (PerkinElmer), allowing for multiparameter phenotyping. Transcriptomic analysis was performed on surgical specimens to assess if differences exist in mRNA expression of tumor-associated and immune genes between pts treated with MK-2206 (n=5) and untreated matched controls (n=5) (nanoString). Statistical analysis was performed using t-Test, NetBID, and multiple comparison analysis by Benjamini-Hochberg. Gene set enrichment analysis (GSEA) was performed within R with gene sets from Molecular Signatures Database (Hallmark, Reactome, GO).
Results: On qmIF analysis, MK-2206 treated pts exhibited a significant increase in median cytotoxic T-cell (CD3+CD8+, CTL) density between pretreatment biopsy and surgical excision specimens, as compared to the control pts (87% vs.0.2%, p &lt; 0.05). Mean macrophage density (CD68+) was numerically lower in surgical specimens of pts who received MK-2206 vs. control pts, although CD68+ infiltration was overall low (p=ns). mRNA expression supports in vivo activity of MK-2206 with lower expression levels of cell cycle, proliferation and anti-apoptotic genes (e.g. CTNNB1, CCND2, BAX) and greater expression of pro-apoptotic genes (e.g. BAD) associated with MK-2206 treatment (raw p-value &lt;0.05). Additionally, greater mRNA copy number of IGF1R, a receptor tyrosine kinase (RTK) previously identified as upregulated in BC in the context of Akt inhibition, was found in post-MK-2206 surgical specimens as compared to control, non-MK-2206 specimens (raw p-value &lt;0.05). MK-2206 was also associated with reduced expression of myeloid markers (e.g. CSF1R, CD163) (raw p-value &lt;0.05). By GSEA, canonical gene sets related to interferon signaling were increased in post-MK-2206 specimens as compared to non-MK-2206 specimens, whereas monocyte chemotaxis genes were decreased in treated pts (adj p-value &lt;0.05). RT-PCR is currently underway to compare biopsy and surgical specimens for a subset of RTK, immune and apoptosis related genes identified above.
Conclusion: mRNA and qmIF analysis suggest that Akt inhibition, may increase interferon signaling, CTL density, and decrease myeloid infiltration. Thus, Akt inhibition may promote a favorable TME. At present, there are both FDA approved and investigational agents that target the PI3K/mTOR pathway. Further investigation is warranted to understand the impact of Akt inhibition on the TME and potential therapeutic implications.
Citation Format: Marks DK, Gartrell RD, Pan Q, El Asmar M, Hart TD, Esancy CL, Lu Y, Yu J, Hibshoosh H, Connolly E, Kalinsky K, Saenger YM. Akt inhibition associated with change in immunophenotype of tumor microenvironment (TME) in breast cancer (BC) [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 P2-03-01.