Abstract 772: Spatial analysis of the DLBCL tumor microenvironment via the novel SignalStarTM multiplex immunohistochemistry assay

Abstract Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, accounting for more than 18, 000 cases each year within the United States. While roughly 60% of patients with DLBCL achieve durable remissions with existing chemotherapies, the prognosis of patients with refractory DLBCL is poor. The immune landscape of DLBCL is complex and further elucidation of the interplay between T cell activation and immunosuppression within the tumor microenvironment (TME) is necessary in order to better stratify patients and predict outcomes for therapy. This complexity, as well as the relevance of the spatial context of the various cell types within the TME necessitate the ability to simultaneously visualize biomarkers and phenotypic markers via multiplex immunohistochemical (mIHC) assays. Here, we construct a 12-plex SignalStar mIHC panel consisting of T cell phenotypic, activation and suppression markers in order to characterize the TME of FFPE DLBCL cores. We visualize immuno-oncology markers in the context of the tumor and myeloid cells, as defined by CD68. In this assay, all 12 antibodies are applied in one primary incubation, followed by the addition of a network of fluorescently-labeled oligonucleotides used to amplify the signal of 4 antibody-oligo conjugates across 3 rounds of imaging. Following amplification and imaging of the first 4 conjugates, the fluorescent signal was enzymatically removed and the process repeated for two more rounds. The three resulting images were then aligned using Visiopharm’s TissuealignTM platoform. Quantitative analysis, including positive cell counts and marker co-localization, was conducted with Visiopharm’s PhenoplexTM. Biomarker expression patterns were also examined using PhenoplexTM. Finally, we correlated these data with patient demographics, disease progression, and treatment responses. Our data demonstrate that the SignalStar mIHC assay is a powerful tool in the elucidation of the complex immunosuppressive TME, enabling researchers to better understand the responses to immunotherapeutic approaches and chemotherapy combination strategies. Citation Format: Jennifer Ziello, Shuling Zhang, Derek Papalegis, Lily Vu, Gabriella Spang, Jean Loebelenz, Arvin Ruiz, Giorgio Inghirami, Sarah Klein. Spatial analysis of the DLBCL tumor microenvironment via the novel SignalStarTM multiplex immunohistochemistry assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 772.