Abstract 1005: Combination T cell receptor immunosequencing and multiplex immunohistochemistry reveal novel insights into the immune response to human pancreatic cancer
Authors: Seo Yongwoo David , Jalikis Florencia , Jiang Xiuyun , Sullivan Kevin M. , Vignali Marissa , Robins Harlan , Pillarisetty Venu G. 
Published: August 2018

Abstract
Background: Despite advancements in therapy, pancreatic ductal adenocarcinoma (PDA) remains an aggressive cancer with high mortality. It is characterized by dense inflammation, including many T cells; however, it is unclear whether these T cells signify true anti-tumor response. In the setting of disappointing results of immunotherapy in PDA, we sought to gain a deeper understanding of the intratumoral T cell response.
Methods: With IRB approval, we obtained archival resected PDA tumors in paraffin embedded blocks from 54 patients and performed T cell receptor (TCR) immunosequencing on extracted DNA. Productive clonality was defined as 1-Pielou's evenness; TCR fraction was calculated from number of observed templates versus housekeeping genes. Multiplex immunohistochemistry (IHC) was performed on slides immediately adjacent to blocks sent for TCR sequencing using the PerkinElmer Vectra system. False H&E images generated from autofluorescence signatures were analyzed by a pathologist to categorize peritumoral stroma versus lymphoid tissue. Image analysis was performed using FlowJo10, turning object fluorescence data into cytometric outputs. Correlation analysis between multiplex and TCR data was performed using Pearson's correlation.
Results: Mean TCR fraction was 0.27, and mean clonality was 0.15 (typical peripheral blood clonality is 0.08). TCR fraction was positively correlated with clonality (R2=0.23, p=0.007), but there was no survival difference between high or low TCR fraction or clonality. 3.1 million individual cells within the peritumoral stroma were phenotyped in total using multiplex IHC; within the stroma, CD4 T cells, CD8 T cells, and macrophages made up on average 4.6%, 6.8%, and 2.0% of all cells analyzed. 35.6% of CD8 T cells were positive for PD-1, while 20.8% of CD4 cells were FOXP3+ regulatory T cells (Treg). TCR fraction correlated positively with infiltration of CD8+ (R2=0.28, p=0.03) and CD68+ (R2=0.27, p=0.03) cells, but not CD4 cells. Only CD8 cells which were PD-1- correlated positively with clonality (R2=0.26, p=0.04). The presence of Treg correlated very strongly with CD8+PD-1+ cells (R2=0.44, p=0.0004).
Conclusion: Here we demonstrate a novel approach to describe the varying immunophenotypic landscape of human PDA depending on the clonal expansion of intratumoral T cells. T cell infiltration appear to be correlated mostly with CD8 T cells as well as the presence of macrophages. Patients with more clonal expansion also had more CD8 cells that lacked the exhaustion marker PD-1, and the presence of regulatory T cells accompanied environments where CD8 cells were positive for PD-1. These data suggest that cytotoxic T cell exhaustion is a phenomenon which may be regulated by Treg and have a negative impact on clonal expansion of intratumoral T cells in human PDA.
Citation Format: Yongwoo David Seo, Florencia Jalikis, Xiuyun Jiang, Kevin M. Sullivan, Marissa Vignali, Harlan Robins, Venu G. Pillarisetty. Combination T cell receptor immunosequencing and multiplex immunohistochemistry reveal novel insights into the immune response to human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1005.