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Abstract
Background Tumour-associated macrophages (TAMs) are key immune cells in the microenvironment of diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma (NHL). The conventional M1/M2 classification of TAMs does not adequately represent macrophage diversity, causing inconsistent prognostic significance in DLBCL. This study used spatial whole-transcriptomic atlas (WTA) analysis to characterise CD68+ cells in distinct anatomical regions of lymphoid germinal centres, the tissue of origin for DLBCL. Macrophage signatures (MacroSigs) derived from these regions were evaluated in clinical cohorts of DLBCL treated with R-CHOP or a novel immunotherapy combination.
Methods Digital spatial profiling (DSP) with WTA analysis of CD68+ cells was performed in 24 reactive lymphoid tissue samples to define MacroSigs representing distinct lymphoid spatial niches (figure 1). Eight DLBCL datasets (4,594 patients) with transcriptomic and survival information were analysed to clinically evaluate the spatial-derived MacroSigs. Comparative T cell studies were conducted using DSP WTA data from CD3+ cells (n=87), projected onto a scRNAseq dataset. The correlation of MacroSigs with the response to T cell targeted therapy in relapsed/refractory NHL was investigated through RNAseq from patients (n=40) treated with Glofitamab and Englumafusp alfa combination in phase 1 trial (NCT04077723).
Results DSP analysis revealed transcriptomics differences between macrophages in distinct spatial compartments of the germinal centre, including light zone (LZ) and dark zone (DZ) regions. The DZ-MacroSig, distinct from the B-cell-derived DZ signature, was prognostic for shorter overall survival in multiple datasets of R-CHOP-treated DLBCL (adjusted P <0.05 in 7/8 datasets) (figure 2).1 Building on this work, we projected the CD3+ transcriptome derived from samples classified as ‘DZ-MacroSig high’ onto a scRNAseq T cell reference map and observed that DZ-macrophage enriched tumours showed a relative enrichment in T-regulatory cells (Treg) (figure 3). We, therefore, tested the DZ-LZ MacroSigs in the setting of T cell directed therapy and demonstrated that patient stratification based on LZ- or DZ-MacroSigs was associated with therapy response in a phase 1 trial of the Glofitamab and Englumafusp alfa combination (figure 4).
Conclusions This study provides spatially-resolved evidence of macrophage diversity between the light and dark zones of the germinal centre, with prognostic relevance in lymphoid tumours. DLBCL patients with DZ-like macrophages have poor outcomes on R-CHOP treatment, while those with LZ-like macrophages show good outcomes with novel immunotherapeutic combinations enhancing T cell activities. These findings may offer approaches to patient stratification for early incorporation of T cell directed therapy in DLBCL.
Acknowledgements M.L. was supported by the China Scholarship Council (202006940018). A.D.J. was supported by the Singapore Ministry of Health’s National Medical Research Council Clinician Scientist Award (MOH-000715-00). Work in A.D.J.’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, and the Italian Ministry of Education, University and Research (MIUR) Grant 2017K7FSYB. GB was supported by Italian Ministry of Education, University and Research (MIUR) through the ‘PON Research and Innovation 2014–2020’.
Reference
Liu M, Bertolazzi G, Sridhar S, et al. Spatially-resolved transcriptomics reveal macrophage heterogeneity and prognostic significance in diffuse large B-cell lymphoma. Nat Commun. 2024;15(1):2113. Published 2024 Mar 8. doi:10.1038/s41467-024-46220-z.
Ethics Approval Our research complies with all relevant ethical regulations. Biopsy samples were pre-treatment samples and obtained from the Department of Pathology, National University Hospital, with IRB approved waiver of consent in accordance with the ethical guidelines of the National Healthcare Group domain specific review board (NHG DSRB) approved protocol 2015/00176. This waiver of consent applies to all samples obtained between 1st January 1990 and 30th April 2020 on the basis that there is no longer patient contact (patient is deceased or lost to follow-up) and that this study poses minimal risk to the patient. A portion of the reactive lymphoid tissue samples were from obtained the archives of the Tumor Immunology Laboratory of the University of Palermo and approved by the University of Palermo Institutional Review Board 09/2018. Data from DLBCL patients treated with with Glofitamab and Englumafusp alfa combination were obtained from phase I clinical trial (NCT04077723).
Schematic of GeoMx® DSP WTA workflow (created with BioRender.com)
Spatially-derived MacroSig3/4 (LZ/DZ) stratify for patient survival in DLBCL datasets
DZ MacroSigs enriched tumours showed a relative enrichment in T-regulatory cells (Treg)
Association between patient stratification based on LZ- or DZ-MacroSigs and therapy response in a phase 1 trial of the Glofitamab and Englumafusp alfa combination (NCT04077723). CMR, Complete metabolic response
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