Abstract B033: Multiplex analysis of the tumor immune microenvironment during treatment with atezolizumab/pelareorep/letrozole reveals novel immune-tumor interactions
Authors: Dadrastoussi Homa , Olea Julian , Hernandez Eduardo Fernandez , Martinez Hugo Lara , Wu Kaijin , Loghmani Houra , Heineman Thomas , Trauger Richard , Coffey Matt , Merchant Akil , Kelly Kevin 
Published: December 2023

Abstract
Background: Pelareorep (pela) is a non-modified intravenously administered oncolytic reovirus exhibiting effective tumor suppression through both innate and adaptive immune responses, as well as direct tumor lysis. Findings from the AWARE-1 window of opportunity study previously demonstrated the synergistic potential of combining pela with atezolizumab (atezo), showcasing promising immunological reactions within tumors of early breast cancer (eBC) patients. To gain deeper insights into the complex tumor immune microenvironment (TiME) pre- and post-treatment, we employed imaging mass cytometry (IMC) to conduct high-dimensional, single-cell analysis of tissue samples.
Methods: Patients (n=10) received pela (days 1, 2 and 8, 9), atezolizumab (day 3), and letrozole (day 1 to 21). Tumor biopsies (FFPE samples) were collected pre-treatment on day 3 (prior to atezolizumab administration) and on day ~21 (surgical excision). Samples from 8 out of 10 patients were subjected to IMC with a comprehensive panel of 37 antibodies for single cell investigation of TiME changes. Tumor, proliferation, T cell, macrophage, NK, and immunoregulatory markers were included in the panel.
Results: Our first pass analysis of IMC images, showed an increase in proliferating (Ki67+) cytotoxic T cells adjacent to apoptotic (caspase 3+) tumor cells post-treatment (both D3 and D21) samples indicating immunogenic cell death. Conversely, we observed a decrease in Ki67+ and ER-positive tumor cells after treatment indicating decreased tumor cell proliferation and hormone expression. In 3 out of 8 patients, treatment was associated with a shift in monocyte/macrophages from an M1 (CD68+/CD163-) to M2 (CD68+/CD163+) phenotype associated with significant tumor infiltration. Surprisingly, in these patients we also noted a significant increase in apoptotic M2 macrophages on D21. Consistent with the known immune priming effects of pela, both PD-1 and PD-L1 increased on D3. Subsequently, on day 21, post atezo, tumor PD-L1 decreased on tumor cells while PD-1 expression persisted. Pela induced IDO expression on both tumor and monocyte/macrophage cells. Unlike its effect on PD-L1, atezo did not appear to attenuate IDO expression on day 21. We were also able to detect a treatment related increase in immune cell perivascular localization on D3 providing further evidence of TiME priming.
Conclusions: The immune system plays a crucial role in regulating cancer progression; however, our understanding of immune interactions with tumors remains limited. Our study utilizing IMC revealed complex immune-tumor interactions changes during treatment with atezo/pela/letrozole. These findings provide valuable insights into the intricate dynamics of the TiME and may inform new therapeutic combination approaches in breast cancer treatment.
Citation Format: Homa Dadrastoussi, Julian Olea, Eduardo Fernandez Hernandez, Hugo Lara Martinez, Kaijin Wu, Houra Loghmani, Thomas Heineman, Richard Trauger, Matt Coffey, Akil Merchant, Kevin Kelly. Multiplex analysis of the tumor immune microenvironment during treatment with atezolizumab/pelareorep/letrozole reveals novel immune-tumor interactions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B033.