Abstract 4063: Single cell immune profiling of patients with advanced biliary cancers treated with combination checkpoint inhibition and GM-CSF reveals diverse T cell and myeloid cell mechanisms of action
Authors: Keenan Bridget P. , Tamaki Whitney , Liu Eric , Chen Brandon , Cheung Alexander , Gordan John D. , Sheldon Brenna , Zhang Li , Kelley Robin K. , Fong Lawrence 
Published: August 2019

Abstract
Background: Advanced biliary cancers (ABC) including cholangiocarcinoma and gallbladder adenocarcinoma are rising in incidence with limited standard treatment options. While checkpoint inhibition (CPI) achieves durable tumor responses in subsets of patients across many malignancies, less than 10% of ABC patients respond to single agent PD-1-targeted therapy. Combination immunotherapy aims to overcome pre-existing and adaptive resistance to immunotherapy. GM-CSF is a cytokine that activates and matures antigen-presenting cells, suggesting the potential to enhance immune responses. The exact mechanisms of action of this cytokine have not been defined in cancer patients. The combination of GM-CSF and anti-CTLA-4 CPI demonstrated safety along with inducing clinical responses in melanoma and prostate cancer. We conducted a phase II trial of the novel combination of GM-CSF and pembrolizumab (Pembro) in patients with ABC which has resulted in durable clinical responses in greater proportion than previously reported with anti-PD-1 monotherapy.
Methods: We assessed peripheral blood mononuclear cells (PBMC) from patients on Pembro monotherapy and combined Pembro/GM-CSF by mass cytometry (CyTOF) and T cell receptor (TCR) sequencing. We explored for the differences between clinical responders versus non-responders.
Results: We find that the addition of GM-CSF to Pembro leads to a higher frequency of myeloid cells overall; however, certain sub-populations of classical monocytes and conventional dendritic cells decreased in peripheral blood following upfront Pembro followed by GM-CSF. GM-CSF did not seem to change the phenotypes or relative frequencies of circulating T cell subsets. We also find that clinical responders possess specific circulating populations of classical monocyte and conventional dendritic cells prior to treatment. Responders also had higher percentages of CD8+ T cells expressing CD39 following Pembro treatment compared to non-responders. Combination therapy with Pembro/GM-CSF led to different effects on the T cell repertoire compared to Pembro alone.
Conclusions: The addition of GM-CSF to Pembro leads to dynamic shifts in myeloid cell subsets in the peripheral blood of ABC patients treated with immunotherapy, whereas Pembro alone led to changes in the activation states of T cell subsets. The TCR repertoire shifts reflect distinct mechanisms of action for Pembro monotherapy versus Pembro/GM-CSF. Future studies will explore the mechanisms driving the increased response rate seen with combination immunotherapy in comparison to Pembro alone in ABC patients, both through the study of peripheral immune responses as well as via immune-profiling of tumors from sequential biopsies of patients on Pembro monotherapy versus combined Pembro/GM-CSF.
Citation Format: Bridget P. Keenan, Whitney Tamaki, Eric Liu, Brandon Chen, Alexander Cheung, John D. Gordan, Brenna Sheldon, Li Zhang, Robin K. Kelley, Lawrence Fong. Single cell immune profiling of patients with advanced biliary cancers treated with combination checkpoint inhibition and GM-CSF reveals diverse T cell and myeloid cell mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4063.