Abstract 975: Single cell RNA sequencing of esophageal adenocarcinoma before and after chemotherapy alone and with pembrolizumab identifies novel tumor immune microenvironment alterations
Authors: Thakkar Prashant V. , Sarkar Sandipto , Zhang Chao , Elmonshed Dina , Futamura Emma , Betel Doron , Shah Manish A. 
Published: September 2020

Abstract
The PD-1 inhibitor, pembrolizumab, was recently approved for PD-L1+ esophageal squamous cell cancer (ESCC) with a response rate (RR) of 14.3-16.7% (KEYNOTE -180, 181). Pembrolizumab is minimally active in esophageal adenocarcinoma (EAC) (RR 5.2%-7.7%, KEYNOTE-180,-181). Biomarkers, such as PD-L1 expression and mutation burden are poor predictors for response to pembrolizumab in EAC. We examined the tumor immune microenvironment (TIME) by single cell RNA sequencing (scRNAseq) in EAC to improve our understanding of local immunity in EAC and how it changes with treatment. Patients with locally advanced EAC (pT2N+ or pT3-4N-/+, M0) underwent endoscopic biopsies of the tumor and adjacent non-tumor tissue (NTA) at baseline and following induction treatment with either paclitaxel/carboplatin (PC) alone or PC + pembrolizumab (NCT02998268 clinicaltrials.gov). These biopsies were subjected to scRNAseq analysis using the 10X genomics platform. scRNAseq data from 65,538 individual cells isolated from 17 baseline endoscopic EAC tumor biopsies revealed dozens of cell populations that largely grouped into 9 different clusters. Across all the baseline tumor samples assessed, epithelial (47.8%), keratinocytes (9.9%), and endothelial cells (9.8%) were the most abundant cell populations, consistent with tumor architecture in vivo. Among the immune cells (20% of the total), B cells (7.1%), T cells (6.9%) and Monocytes (2.2%) were the most abundant tumor-associated cell populations.
We further assessed matched baseline and on-treatment biopsies (n=10) from six patients that received PC and four patients that received PC + pembrolizumab. We saw a significant reduction in tumor epithelial cells upon treatment in both arms suggesting effectiveness of treatment (P&lt;0.001). PC alone significantly induced tumor-associated B cells (P≤0.0007) and T cells (P≤0.002) with treatment. In contrast, treatment with pembrolizumab + PC specifically was associated with significant increase in tumor-associated neutrophils (P&lt;0.001) and dendritic cells (P≤0.007). Interestingly, comparing across PC only vs PC + Pembrolizumab, we see an increase in number of B cells (P≤0.020), neutrophils (P&lt;0.001) and NK cells (P&lt;0.001) with addition of Pembrolizumab. Furthermore, specific immune cell subsets including CD4+ Th17, Th2, Treg and CD8+ stimulated cells show significant associations with treatment regimens.
Our study demonstrates that scRNAseq provides a rich description of the TIME with significant value for clinical trials. The TIME of EAC is complex, largely immunosuppressive, and chemotherapy with, or without, pembrolizumab is associated with significant unique alterations in TIME. We are currently investigating differences in TIME composition between tumor and NTAs to better understand the components that contribute to tumor growth.
Citation Format: Prashant V. Thakkar, Sandipto Sarkar, Chao Zhang, Dina Elmonshed, Emma Futamura, Doron Betel, Manish A. Shah. Single cell RNA sequencing of esophageal adenocarcinoma before and after chemotherapy alone and with pembrolizumab identifies novel tumor immune microenvironment alterations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 975.