SPATIALLY‐RESOLVED TRANSCRIPTOMICS DEFINE CLINICALLY RELEVANT SUBSETS OF MACROPHAGES IN DIFFUSE LARGE B‐CELL LYMPHOMA

Abstract

Introduction: Tumor-associated macrophages (TAMs) are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL) and are implicated in tumor progression and therapy resistance. Conventional immunohistochemistry-based studies have relied on the oversimplified M1/M2 classification of TAMs which does not fully capture the functional diversity of macrophage biology, thus potentially contributing to their inconsistent prognostic significance in DLBCL. Spatial whole-transcriptomic analysis allows in-depth investigation of specific cell types in different spatial regions within normal and malignant lymphoid tissues, enabling detailed macrophage phenotyping. Here, we employed spatial whole-transcriptomic analysis in the characterization of CD68+ cells of DLBCL and reactive lymphoid tissues (RLTs) to identify novel macrophage subsets with biological and clinical significance. Methods: Digital spatial profiling with whole-transcriptomic analysis of CD68+ cells was performed in 47 DLBCL and 17 RLTs, to define macrophage signatures (termed “MacroSigs”) of distinct lymphoid spatial niches and clinical scenarios. Eight independent DLBCL datasets (4594 patients) with complete transcriptomic and survival information were used for validation of these spatial-derived MacroSigs. Results: Digital spatial profiling revealed previously unrecognized transcriptomic differences between macrophages populating distinct spatial compartments in RLTs (light zone (LZ)/dark zone (DZ), germinal center (GC)/interfollicular (IF) regions), and in between disease states (RLTs and DLBCL with or without relapsed disease). This transcriptomic diversity of macrophages was categorized into eight MacroSigs. Spatial-derived MacroSigs associate with specific cell-of-origin (COO) subtypes of DLBCL, of particular interest being the IF-MacroSig enriched in the unclassified COO (p < 0.005, 6/8 datasets). MacroSigs of relapsed-DLBCL and DZ were prognostic for shorter overall survival in multiple datasets (p < 0.05 in 5/8 datasets; p < 0.05 in 8/8 datasets, respectively). Projection onto a macrophage single-cell RNA-sequencing atlas reveals the Non-relapse-DLBCL MacroSig to depict HES1/FOLR2-like macrophages, while relapse-DLBCL-MacroSig represents IL1B-like monocytes, with unique therapeutic vulnerabilities for each. The research was funded by: Work in ADJ’s laboratory is funded by a core grant from the Cancer Science Institute of Singapore, National University of Singapore through the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative. CT was supported by the Italian Foundation for Cancer Research (AIRC) Investigator Grant IG ID.22145; 5 × 1000 Grant ID.22759. Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.