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Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4 + helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1 + CXCL13 + T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46% of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5 + normal B cells in close proximity to CXCL13 + T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1 + CXCL13 + T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL ( P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1 + CXCL13 + T cells as a treatment target in LR-CHL., Competing Interests: Competing interest statement: C.S. has performed consultancy for Seattle Genetics, Curis Inc., Roche, AbbVie, Juno Therapeutics, and Bayer and has received research funding from Bristol-Myers Squibb, Epizyme, and Trillium Therapeutics Inc. C.S. and D.W.S. are coinventors on a patent (“Method for determining lymphoma type”) using NanoString technology. D.W.S. has performed consultancy for Abbvie, AstraZeneca, Celgene, and Janssen and has received research funding from Janssen, NanoString, and Roche., (Copyright © 2021 the Author(s). Published by PNAS.)

The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, were analyzed as a discovery cohort, followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) exhibited a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (n = 18) had a significantly distinct pattern that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV- GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas., (© 2021 by The American Society of Hematology.)

Gray zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classic Hodgkin lymphoma (cHL), is a rare and poorly defined entity. Alongside GZL, a subset of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) has been described with polymorphic/GZL-like morphology (polymorphic-EBV-L). To fill the important gap in our understanding of the pathogenic process underlying these entities, we performed a gene expression study of a large international cohort of GZL and polymorphic-EBV-L, combined with cHL and primary mediastinal large B-cell lymphoma (PMBCL) cases. In an unsupervised principal component analysis, GZL cases presented with intermediate scores in a spectrum between cHL and PMBCL, whereas polymorphic-EBV-L clustered distinctly. The main biological pathways underlying the GZL spectrum were related to cell cycle, reflecting tumor cell content, and extracellular matrix signatures related to the cellular tumor microenvironment. Differential expression analysis and phenotypic characterization of the tumor microenvironment highlighted the predominance of regulatory macrophages in GZL compared with cHL and PMBCL. Two distinct subtypes of GZL were distinguishable that were phenotypically reminiscent of PMBCL and DLBCL, and we observed an association of PMBCL-type GZL with clinical presentation in the "thymic" anatomic niche. In summary, gene expression profiling (GEP) enabled us to add precision to the GZL spectrum, describe the biological distinction compared with polymorphic-EBV-L, and distinguish cases with and without thymic involvement as 2 subgroups of GZL, namely PMBCL-like and DLBCL-like GZL., (© 2020 by The American Society of Hematology.)

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma-specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3 + T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3 + T cells in the direct vicinity of MHC class II-deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell-like immunosuppressive subset of LAG3 + T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints. See related commentary by Fisher and Oh, p. 342 . This article is highlighted in the In This Issue feature, p. 327 ., (©2019 American Association for Cancer Research.)