Abstract 2789: Highly multiplexed imaging mass cytometry reveals immune cell composition and spatial heterogeneity in diffuse large B cell lymphoma associated with treatment outcome

Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogenous entity characterized by its variable clinical and biological behaviour, and approximately 30% of patients experience relapsed or refractory disease after first-line therapy. We hypothesize that a better characterization of the tumor microenvironment (TME) might help identify patients who may benefit from individualized immunotherapies. Similar studies in this area have been limited by technical challenges - conventional highly multiplexed techniques require tissue disruptions that lose spatial information, while those that retain tissue architecture can only examine 6-8 markers simultaneously. We and others have previously reported that PD-L1 expression is correlated with decreased survival in a cohort of 85 DLBCL patients. In the present study, we characterized TME components, including their types, frequency and spatial interaction, in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution. Using a panel of 32 antibodies, IMC was performed on a subset of our previously studied cohort. We examined 41 cores from 33 DLBCL cases, 17 GCB and 16 non-GCB, by Hans criteria. Clinical outcome data were available for 29 patients, 22 of whom had complete response (CR) to R-CHOP therapy while 7 had primary refractory disease. Using both supervised gating and unsupervised clustering algorithm, IMC data were analyzed for relevant immunophenotypes and compared across clinical outcome groups. The TME was mainly composed of 13.1% ± 1.9% (mean ± SE) CD4+ T-helper cells, 10.8% ± 1.1% CD8+ cytotoxic T cells, 6.3% ± 0.9% CD68+ macrophages, 2.7% ± 0.5% FoxP3+ regulatory T cells, and 58.1% ± 3.4% tumor cells. In non-GCB group, higher ratio of regulatory T cells was associated with refractory disease. In contrast, activated granzyme-B+/CD8+ cytotoxic T cells were more frequent in CR group, while markers of exhaustion (Tim3, Lag3) were found in patients with refractory disease. To gain functional insight into the various immune subsets, we performed spatial analysis of the immune cells and their relation to blood vessels and tumor cells. Nearest distance analysis showed that CD4+ cells were most tightly clustered around blood vessels in patients with CR, while in those with refractory diseases, CD4+ cells were further away from the vessels (p=0.03). On the contrary, distances between cytotoxic T cells and regulatory