Abstract B51: Quantitative multiplex immunofluorescence reveals that chemoradiation therapy favorably modulates the tumor immune microenvironment of pancreatic cancer
Authors: Enzler Thomas , Gartrell Robyn , Fazlollahi Ladan , Perni Subha , Kim Pan , Hart Thomas , Monsalve Christian , Green Samuel , Saenger Yvonne , Horowitz David P. 
Published: February 2019

Abstract
Immunotherapy has altered the therapeutic landscape in several malignancies but to date has not been effective in treatment of pancreatic ductal adenocarcinoma (PDAC). Patients with advanced PDAC continue to have median survival under one year and thus new therapeutic approaches for this daunting disease are urgently needed. It is well known that radiation therapy causes the release of tumor antigens and pro-inflammatory cytokines leading to stimulation of anti-tumor immunity. In our study, we sought to quantify this effect by comparing immune cell infiltration in tissues of PDAC treated with chemoradiation to untreated tumors.
Slides obtained from surgical resection specimens were stained using quantitative multiplex immunofluorescence (qmIF) for: CD3, CD4, CD8, FOXP3, CD68, and Ki-67. Nuclei were labeled using DAPI. Slides with tumor tissue were then imaged (15-20 images per slide) using automated imaging system VECTRA and they were analyzed using inForm software to evaluate cell density in tumor and stroma compartments. Overall, slides were obtained and analyzed from 6 different patients who underwent chemoradiation therapy and from 5 patients who did not receive neoadjuvant treatment.
When comparing treated vs. untreated tumors, CD3+ densities were significantly higher in the stromal tissue of the treated tumors vs. untreated tumors (P = 0.0095). CD3+CD4+FOXP3- T helper cells were similarly significantly higher in treated tumors (P = 0.0095), while there was a trend towards increase in CD3+CD8+ cytotoxic T cells (P = 0.0667). Conversely, CD4+FOXP3+ cells were increased in untreated tumors although significance was not reached (P = 0.15). No clear differences in infiltrating CD68+ macrophages were observed between treated and untreated tumors.
To our knowledge this is the first time that immune cell infiltrates of PDAC were characterized using qmIF. The higher counts of CD3+CD4+ T helper cells and of CD3+CD8+ cytotoxic T cells found in tumors treated with chemoradiation can be explained by the increased immunogenicity caused by treatment. A tendency towards a higher density of CD4+FOXP3+ cells in untreated tumors may contribute to immunosuppression in the tumor microenvironment as there were less CD3+CD8+ cytotoxic T cells and CD3+CD4+ T helper cells found in those tumors.
We are in the process of collecting and staining additional samples to build on these findings. We will correlate our data with survival data to identify biomarkers predictive of outcome and provide rationale for the development of new treatment strategies for this challenging disease.
Citation Format: Thomas Enzler, Robyn Gartrell, Ladan Fazlollahi, Subha Perni, Pan Kim, Thomas Hart, Christian Monsalve, Samuel Green, Yvonne Saenger, David P. Horowitz. Quantitative multiplex immunofluorescence reveals that chemoradiation therapy favorably modulates the tumor immune microenvironment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B51.