Background Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Conventional immunohistochemistry-based studies with varying prognostic significance precludes a comprehensive analysis of macrophage subtypes in DLBCL. We hypothesized that whole-transcriptomic analysis (WTA) of macrophage in-situ would identify new macrophage subsets of biological and clinical significances. Methods Digital spatial profiling with WTA of CD68+ cells was performed in 47 DLBCL and 17 reactive lymphoid tissues (RLTs), to define macrophage signatures (termed 'MacroSigs') of distinct lymphoid spatial niches and clinical scenarios. Eight independent DLBCL datasets (4,594 patients) with transcriptomic and survival information were used for validation of 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-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 (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. Conclusions This study first provides spatially-resolved macrophage WTA in reactive and malignant lymphoid tissues. Gene expression signatures of macrophages in the DZ and relapsed-DLBCL samples are consistently prognostic in multiple datasets and offer insights into novel therapeutic strategies for DLBCL.

The authors have declared no competing interest.

ML was supported by the China Scholarship Council (202006940018). ADJ was supported by the Singapore Ministry of Health National Medical Research Council Clinician Scientist Award (MOH-000715-00). The study 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; 5x1000 Grant ID.22759.

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All biopsy samples were obtained from the Department of Pathology, National University Hospital, in accordance with the ethical guidelines of the domain specific review board (DSRB) approved protocol 2015/00176.

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All data produced in the present study are available upon reasonable request to the authors.