Abstract 3102A: Analysis of spatial relationships between infiltrating immune cells within the tumor microenvironment following combinatorial immunotherapy
Authors: Thomas Dwayne L. , Murphy Adrian G. , Weiss Matthew J. , He Jin , Makary Martin A. , Burkhart Richard A. , Wolfgang Christopher L. , Jaffee Elizabeth M. , Zheng Lei , Thompson Elizabeth D. 
Published: August 2019

Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy, which is the third leading cause of cancer related deaths in the United Sates. As the number of new cases continues to rise, the current treatments of surgical resection, chemotherapy, and radiation have not drastically improved PDAC survival rates. PDAC is known to have a quiescent immune microenvironment, which is resistant to single-agent checkpoint inhibitors. We identified a combination approach using immune checkpoint inhibition and immune activing agonists to prime the tumor microenvironment (TME) to activate T-cells in order to overcome this “cold” environment. Here we investigate spatial relationships between CD8+, PD-1+ (programmed cell death protein), and PD-L1+ (programmed cell death ligand) cells in the TME following single agent and combinatorial immunotherapy protocols.
Experimental Procedures: Our cohort includes 17 surgically resected patient tumor specimens from formalin-fixed paraffin-embedded tissue blocks. These patients either received GM-CSF vaccine (GVAX) with or without immune checkpoint inhibitor, anti-PD-1. Of the 11 patients analyzed, six received GAVX with anti-PD-1 while the others received GVAX alone. Automated immunohistochemical staining was performed for CD8, PD-1, and PD-L1. Analysis was performed using HALO image analysis software (Indica Labs) (HALO v2.0.1145.31). Proximal distance (Nearest Neighbor Analysis) of CD8+ to PD-1+ cells, CD8+ to PD-L1+ cells, PD-1+ to PD-L1+ cells within the tumor area was measured for each patient to generate average distance between cell types.
Results: Of the 17 PDAC patients in our cohort, 11 patient specimens were analyzed. Nearest Neighbor Analysis between the two groups shows there is a trend of increased distance between CD8+ cells and PD-1+ cells and between CD8+ cells and PD-L1+ cells in patients receiving GVAX and anti-PD-1 compared to GVAX alone (p=0.032 and n.s., respectively). Proximal distance analysis of PD-1+ cells to PD-L1+ cells is ongoing.
Conclusions: PDAC remains a considerable diagnostic and therapeutic challenge due to its poor survival outcomes. However, immunotherapy may provide an alternative strategy to improve survival for PDAC patients. Our results suggest that monotherapy with GVAX versus combination therapy with GVAX plus anti-PD-L1 leads to differences in the spatial relationship between CD8+ cells and PD-1+ and PD-L1+ cells, specifically, greater distance between CD8+ T cells and PD-L1+ and PD-1+ cells, suggesting the potential for greater functionality and less direct cell/cell suppression. More broadly, our results demonstrate the ability to detect differences in patient groups based on spatial analysis of distance between cell types, a technique that can be applied generally to the correlative analyses of immunotherapy protocols.. Further analysis of spatial relationships and immune cell infiltration in the TME following immunotherapy will help elucidate how these treatment protocols impact the architecture of the TME and will help guide therapeutic approaches to immunotherapy in pancreatic cancer. We demonstrate a technique of spatial analysis that will aid in dissecting these cellular relationships.
Citation Format: Dwayne L. Thomas II, Adrian G. Murphy, Matthew J. Weiss, Jin He, Martin A. Makary, Richard A. Burkhart, Christopher L. Wolfgang, Elizabeth M. Jaffee, Lei Zheng, Elizabeth D. Thompson. Analysis of spatial relationships between infiltrating immune cells within the tumor microenvironment following combinatorial immunotherapy [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 3102A.