Abstract 3648: The spatial organization of cells expressing MYC and BCL2 affects immune microenvironment composition and prognosis in DLBCL
Authors: Shridar Shruti , Hoppe Michal Marek , Jaynes Patrick , Kumar Gayatri , Jasoria Siddham , Meng Ziwei , Rajan Vaibhav , Kannan Kasthuri , Tripodo Claudio , Jeyasekharan Anand Devaprasath 
Published: March 2024

Abstract
Point process analyses are widely used in ecology and geography to understand the impact and relevance of spatial distribution of points in a complex system. To date, only few studies have explored point process analyses in the context of tumor heterogeneity. Malignancies often show variable patterns of cellular distribution, and the relationship of these topographic variables with underlying biological processes and clinical outcomes is not well understood. Here, we performed spatial point process analyses in diffuse large B-cell lymphoma (DLBCL), leveraging advances in multiplexed immunohistochemistry and cellular phenotyping, with downstream multi-omic and clinicopathological analyses.
We have demonstrated that survival in DLBCL is strongly associated with the fraction of malignant cells showing co-expression of the oncogenes MYC and BCL2 in the absence of BCL6 (M+2+6-). These cells display non-random spatial organization within tumor infiltrates, deviating from a poisson distribution. We therefore modeled the spatial organization of M+2+6- cells within DLBCL, using x-y coordinate information from multiplexed fluorescent immunohistochemistry (mfIHC) in four independent cohorts (N=449 patients). We derived spatial point patterns, upon which Geyers point process models were applied. We observed that patients could be divided into two groups based on these: one group showed “clustered” spatial organization, while the other displayed a “dispersed” M+2+6- cell distribution. Cases with predominantly “dispersed” M+2+6- cells showed shorter overall survival in all analyzed cohorts (P &lt; 0.05 in 4/4 cohorts).
We performed multi-omic analyses to identify potential biological explanations for this association between topography and clinical outcome. Using digital spatial profiling of the transcriptome from CD20, CD3 and CD68 compartments in 47 DLBCL samples, we observed that lymphomas with an M+2+6- ‘dispersed’ phenotype had an immunologically cold microenvironment enriched in Tregs and exhausted CD4+ and CD8+ T cells. Through an integration with single-cell transcriptomic analyses (N=22 samples) we observed that malignant B-cells from cases with M+2+6- ‘dispersed’ phenotype expressed genes implicated in cell migration and adhesion, but also in potentially targetable immune checkpoints such as LAG3.
In summary, by integrating multiplexed immunophenotyping and point process analysis with molecular profiling in the setting of DLBCL, we show that a dispersed pattern of distribution of the M+2+6- subclonal fraction uniquely confers poor prognosis, and associates with a potentially reversible immune cold microenvironment composition. This represents the first demonstration that the spatial distribution of malignant cell subpopulations in lymphoma can embody biological and clinical significance.
Citation Format: Shruti Shridar, Michal Marek Hoppe, Patrick Jaynes, Gayatri Kumar, Siddham Jasoria, Ziwei Meng, Vaibhav Rajan, Kasthuri Kannan, Claudio Tripodo, Anand Devaprasath Jeyasekharan. The spatial organization of cells expressing MYC and BCL2 affects immune microenvironment composition and prognosis in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3648.