Abstract 2254: Immunotopography characterization after neoadjuvant therapy in the pancreatic cancer microenvironment
Authors: Reyes Carmen Mota , Kiessler Maximilian , Bordignon Pino , Aubert Samuel , Pulimeno Pamela , Friess Helmut , Istvanffy Rouzanna , Demir Ihsan Ekin 
Published: April 2023

Abstract
Pancreatic cancer is one of the most common cancers worldwide and its incidence is increasing globally. Treatment consists of surgical resection followed by adjuvant or neoadjuvant therapy. Neoadjuvant therapy (neoTx) has dramatically improved the prognosis of patients with locally advanced and borderline resectable pancreatic cancer. NeoTX has been shown to reverse the immunosuppression exerted by malignant cells and selectively deplete regulatory T cells and myeloid-derived suppressor cells in the peritumoral niche. In addition, neoTx mediates a significant decrease in the severity of neural invasion and stroma activation. However, the effect of neoTx on the topographical interactions between the different populations of tumor-infiltrating immune cells, the degree of intratumoral immune infiltration and distance to tumor cells as well as their spatial interactions with other key features of the tumor microenvironment (TME) remained unknown. In this study, we employed a multiplex immunofluorescence approach on the COMET™ platform from Lunaphore. COMET™ performs full-automated sequential immunofluorescence (seqIF™) assays, which consist of cycles of staining, imaging, and elution. We developed a customized 12-plex panel on COMET™ to characterize the TME of paraffin-embedded sections of pancreatic cancer samples. COMET™ outputs are OME.TIFF files that were analyzed and quantified with QuPath and CellProfiler for cell segmentation and cell phenotyping respectively. The optimized panel was used to analyze the immune architecture of neoadjuvant-treated pancreatic cancer patients compared to primary resected ones who underwent surgical resection with curative intention. We detected the following immune phenotypes: cytotoxic T cells (CD8+, CD3+), helper T cells (CD4+, CD3+), B cells (CD20+, CD3-), regulatory T cells (FoxP3+, CD4+), macrophages (CD68+), neutrophils (CD163+, CD11b+), and myeloid-derived suppressor cells (CD163, CD11b+, CD66b+). We also quantified the presence of both immune checkpoints (PD1+) cells and their apoptotic rate (Caspase-3), and how those correlate with the immunotopography after neoadjuvant therapy. Our optimized 12-plex panel was successfully transferred to an initial cohort of 10 patients. Our preliminary analysis shows interesting results on the effect of neoTx treatment on the immune microenvironment, such as decreased immune cell density, induction of apoptosis in lymphoid cells, and increased PD1 expression in T-killer cells compared to samples from primary resected patients. Our approach demonstrates the importance of a deep topographic characterization to understand the TME composition of pancreatic cancers, with or without neoTx treatment. Our preliminary findings highlight new differential cell identities in neoTx-treated patients, confirming their suitability for application in future clinical immunotherapy trials.
Citation Format: Carmen Mota Reyes, Maximilian Kiessler, Pino Bordignon, Samuel Aubert, Pamela Pulimeno, Helmut Friess, Rouzanna Istvanffy, Ihsan Ekin Demir. Immunotopography characterization after neoadjuvant therapy in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2254.