Citations

Abstract Recent single-cell transcriptomic studies of follicular lymphoma (FL) have revealed heterogeneity among malignant B (MB) cells and selected components of the tumor microenvironment (TME), but have been constrained by relatively small sample sizes and a lack of spatial context. We performed comprehensive cellular profiling and spatial dissection of over five million cells by integrating single-nucleus RNA sequencing (snRNA-seq) from 182 samples (FL, n = 167; control, n = 15) with single-cell spatial transcriptomics (ST) from 386 samples (FL, n = 365; control, n = 21). After stringent quality control, 1,289,073 and 3,968,427 cells from snRNA-seq and ST data, respectively, were analyzed. Unsupervised clustering of snRNA-seq data identified 58 TME cell subtypes. To extend this classification to the ST dataset, we integrated snRNA-seq and ST data by co-embedding them into a shared principal component space, enabling robust batch correction and cell-type label transfer across modalities. Using this supervised annotation framework, ST data recapitulated 54 TME cell subtypes. MB, proliferating MB (MBprolif), and healthy B (HB) cells were classified using sample-level unsupervised clustering in ST. Macrophages exhibited previously uncharacterized heterogeneity in transcriptomic profiles and spatial distribution. We identified CXCL13-expressing macrophage subtypes with high intra- and peri-follicular abundance, termed follicular macrophages (FMs). Non-negative matrix factorization of intrafollicular cell fractions identified five TME archetypes: FM, follicular stroma, two follicular T, and HB archetypes. The FM archetype was enriched for intrafollicular macrophages, including FMs, and showed a significantly higher proportion of grade 3A/3B cases (53.4%) compared to other archetypes (≤16.5%). It was also associated with a higher frequency of high tumor burden requiring treatment initiation rather than a watch-and-wait (WW) approach. Among patients managed by WW, those with the FM archetype showed significantly shorter event-free survival (EFS) compared to those with non-FM archetypes, whereas this association was not observed among treated patients. The percentage of intrafollicular macrophages (IFMP) showed excellent predictive performance for the FM archetype (area under the curve: 0.90). IFMP calculated in a published multiplex spatial protein imaging dataset from an independent cohort of 242 FL patients validated the associations between high IFMP and both pathology grade 3A/3B and shorter EFS in WW patients. Consistently, spatial neighborhood and cell–cell communication analyses revealed co-localization of FMs and MBprolif cells, forming a distinct niche with activated signaling mediated by FM-derived CXCL13, APRIL, and BAFF. Our integrative multi-modal analysis defines previously unrecognized cellular heterogeneity and spatial architecture in FL and highlights macrophage-enriched follicular ecosystems associated with aggressive clinicopathological features, providing a framework for improved biological understanding and patient management. Citation Format: Yoshiaki Abe, Bijal Thakkar, Atish Kizhakeyil, Ashley Wilson, Andrew L. Feldman, Jared Henderson, Amy Ayers, Sara Borgschatz, R. Andrew Harkins, Priya Lakra, Daisuke Kaji, Jonathon B. Cohen, David Russler-Germain, Eric Mou, Francisco Vega, Jennifer Chapman, Giorgio Inghirami, Carla Casulo, Izidore S. Lossos, Jean L. Koff, Chijioke Nze, Peter Martin, Sergei Syrbu, Kiran Vij, David L. Jaye, James R. Cerhan, Christopher R. Flowers, Anne J. Novak, Richard Burack, Dai Chihara, Mamiko Sakata-Yanagimoto, Joshua Tobin, Xubin Li, Michael R. Green. Spatially resolved microenvironment profiling reveals distinct follicular lymphoma archetypes [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A049.

Abstract Few prospective benchmark studies exist to characterize the evolving contemporary real-world practice for peripheral T-cell lymphoma (PTCL). We report the patterns of first-line care and outcomes for 720 patients with systemic PTCL enrolled in 2 related prospective cohort studies, Lymphoma Epidemiology of Outcomes (LEO) from 2015 to 2020 and Molecular Epidemiology Resource (MER) from 2002 to 2015, both followed to 2024. The primary end points were event-free survival (EFS) and overall survival (OS) using Kaplan-Meier estimator and Cox regression model. Secondary end points included correlations of clinical and treatment factors with survival. The most common induction regimens were CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) based (70%), given as CHOP (36%), CHOP plus etoposide (23%), or CHOP-like plus novel agents (11.5%, including 5% BV-CHP [brentuximab plus cyclophosphamide, doxorubicin, and prednisone]). Consolidative autologous stem cell transplant was performed in 102 patients (14%). Within nodal PTCL, EFS and OS were adversely associated with International Prognostic Index 2 to 5, prognostic index for T-cell lymphoma score 1 to 4, and non–anaplastic large-cell lymphoma (ALCL) subtypes. Within LEO, which captured increasing first-line etoposide and brentuximab vedotin, adding etoposide to CHOP was associated with better OS in anaplastic lymphoma kinase-negative ALCL. BV-CHP showed a trend toward OS improvement in ALCL. Patients failing EFS6 and EFS24 had 5-year subsequent OS of 12% and 17%, respectively. The inferior outcomes in non-ALCL subtypes and patients failing EFS6 and EFS24 highlight unmet needs with CHOP-based induction, where clinical trials with targeted therapy should be prioritized. This trial was registered at www.clinicaltrials.gov as NCT02736357.

Abstract Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics. Current classification incorporates clinical presentation and anaplastic lymphoma kinase (ALK) status but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities. We studied 689 patients with ALCL in the LLMPP (Lymphoma/Leukemia Molecular Profiling Project) and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple negative), and immunohistochemistry for phosphorylated STAT3Tyr705. RNA sequencing with unsupervised gene expression profiling in 393 patients identified 2 main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (type I) or absence (type II) of phosphorylated STAT3Y705 expression. Type I ALCLs included ALK+ ALCL and a subset of triple-negative ALCLs (TN-I); type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II). Type I ALCLs were enriched for JAK-STAT3, whereas type II ALCLs were enriched for non–tyrosine kinase pathways, particularly epigenetic regulators such as EZH2. Immunohistochemistry showed overexpression of EZH2 and its trimethylated substrate H3K27. Prognosis in systemic ALCL was favorable for DUSP22-rearranged ALCL (5-year overall survival, 95%) and ALK+ ALCL (88%), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%), and poor for TP63-rearranged ALCL (0%). We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies 4 molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II). This classification can be easily implemented on paraffin tissue in routine practice or clinical trials, and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

Peripheral T cell lymphoma (PTCL) is a heterogeneous group of postthymic T cell neoplasms, with ~40% classified as PTCL–not otherwise specified (PTCL-NOS). PTCL-GATA3, a molecularly defined subtype, associated with T helper 2 (T H 2)–like differentiation and poor prognosis, has frequent co-occurrence of TP53 loss/mutation and heterozygous PTEN loss. CD4+ T cell conditional mouse models with Trp53 mutation/deletion and Pten loss demonstrated mature T cell lymphomas (mTCLs) with T H 2-like transcriptomic and immunophenotypic profiles. Molecular studies revealed that codeletion of Trp53/Pten induced T cell receptor and Janus kinase–signal transducer and activator of transcription signaling, promoting T H 2 differentiation while inhibiting T H 1 differentiation. These findings were validated by CRISPR editing of TP53/PTEN loss in human CD4+ T cells and mechanistically evaluated the p53 binding region in intron-3 of GATA3, resulting in transcriptional repression. Transcriptomic profiles of m-TCLs recapitulated human-PTCL-GATA3 transcriptome and distinguished PTCL-NOS subtypes. Preclinical assessment of m-TCLs with PI3Kγ/δ inhibitors significantly improved survival, supporting a therapeutic approach for the p53-aberrant PTCL-GATA3.

Abstract Molecular characterization of high-grade B-cell lymphoma, not otherwise specified (HGBCL-NOS), is hindered by its rarity, evolving definition, and poor diagnostic reproducibility. To address this challenge, we analyzed 92 HGBCL-NOS tumors collected across Lymphoma/Leukemia Molecular Profiling Project sites. Leveraging comparison cohorts of diffuse large B-cell lymphoma, NOS (DLBCL-NOS) and Burkitt lymphoma (BL), and molecular frameworks described in these entities, our analysis revealed a heterogenous molecular landscape, reminiscent of DLBCL-NOS but with an enrichment of BL features. By cell-of-origin classification, 59% were germinal center B-cell–like (GCB), and 25% were activated B-cell–like (ABC). LymphGen, a genetic classifier for DLBCL-NOS, assigned a genetic subtype to 34% of HGBCL-NOS. Although classification rate was lower than in DLBCL-NOS (66%), assigned subtypes spanned the spectrum of LymphGen classes, including 31% of ABCs classified as MCD. Features differentiating HGBCL-NOS from DLBCL-NOS included MYC rearrangement (47% vs 6%); dark zone signature (DZsig) expression (45% vs 7%); and more frequent mutation of ID3, MYC, CCND3, and TP53, all common to BL. A genetic classifier that differentiates DLBCL-NOS from BL classified 53% of DZsig+ tumors as BL-like, and those classified as DLBCL-like were frequently BCL2-rearranged. Among DZsig− GCB tumors, 95% were DLBCL-like. Centralized pathology review reclassified almost half of tumors as DLBCL-NOS but did not identify a more homogenous HGBCL-NOS population, with no difference in features between confirmed and reclassified tumors. In conclusion, molecular testing enables a subset of HGBCL-NOS to be assigned to established categories. Based on rarity and diagnostic challenges, broader inclusion of HGBCL-NOS should be considered in biomarker-driven DLBCL trials.

ABSTRACTNodal follicular helper T‐cell (T FH) lymphoma of the angioimmunoblastic (AITL) subtype has a dismal prognosis. Using whole‐exome sequencing (n = 124), transcriptomic (n = 78), and methylation (n = 40) analysis, we identified recurrent mutations in known epigenetic drivers (TET2, DNMT3A, IDH2 R172 ) and novel ones (TET3, KMT2D). TET2, IDH2 R172 , DNMT3A co‐mutated AITLs had poor prognosis (p < 0.0001). Genes regulating T‐cell receptor (TCR) signaling (CD28, PLCG1, VAV1, FYN) or activation (RHOA G17V ) or regulators of the PI3K‐pathway (PIK(3)C members, PTEN, PHLPP1, PHLPP2) were mutated. CD28 mutation/fusion was associated with poor prognosis (p = 0.02). WES of purified, neoplastic T‐cell (CD3+PD1+) demonstrated high concordance with whole tumor biopsies and validated the presence of TET2 and DNMT3A in tumor and non‐lymphoid cells, but other mutations (CD28, RHOA G17V , IDH2 R172 , PLCG1) in neoplastic cells. Integrated DNA‐methylation and mRNA expression analysis revealed epigenetic alterations in genes regulating TCR, cytokines, PI3K‐signaling, and apoptosis. RNA‐seq analysis identified fusion transcripts regulating TCR‐activation (8%), revealed a restricted TCR‐repertoire (α = 87%, β = 72%), and showed the presence of Epstein–Barr virus transcriptome (73%). GEP demonstrated the association of B‐cells or dendritic cells in the tumor milieu with prognosis (p < 0.01). RNA‐seq and WES analysis of 12 AITL‐patient‐derived‐xenografts (PDX) showed that bi‐allelic TET2 and DNMT3A mutations or sub‐clonal mutations (PLCG1, PHLPP2) propagated in sequential passages, and gene signatures related to T FH and T CM (central‐memory) were well‐maintained through passages. Gene expression signatures associated with late PDX passages (3rd–5th) were enriched with proliferation and metabolic reprogramming‐related genes and predicted prognosis in an independent AITL series. Low PHLPP2 mRNA expression predicted poor prognosis (p = 0.05) and engineered PHLPP2 or TET2 loss in CD4+ T‐cells showed enhanced PI(3)K activation, thus uncovering a therapeutic target for clinical trials.

Background: Anaplastic large cell lymphomas (ALCLs) represent a heterogeneous group of T-cell lymphomas that currently are classified by the presence or absence of ALK tyrosine kinase (TK) fusion genes (ALK+ or ALK−) and clinical presentation (systemic, cutaneous, or breast implant-associated). Two overarching molecular types of ALCL recently were discovered, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. Fusions involving non-ALK TK genes occur in some ALK− cases, but the fusion landscape of ALCL remains incompletely characterized. Methods: Expert consensus pathology review was conducted in the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). RNAseq was performed and fusions were identified using FusionCatcher. Here, we focused on recurrent in-frame coding fusions. Previously unreported fusions were validated by RT-PCR. Type I/II was assigned using a previously validated gene expression-based model. Genes with adjusted (adj) P&lt;0.05 were considered differentially expressed. Overall survival (OS) was assessed for systemic ALCL, when available. Results: We evaluated 379 ALCLs (229M/150F; mean age, 56 y). Of 199 candidate fusions (excluding reciprocal events), 28 were recurrent and passed quality metrics. At least 1 of these 28 fusions was present in 150 cases (40%). ALK fusions were present in 106 ALK+ ALCLs (all Type I; P&lt;0.0001). Of these, NPM1::ALK was seen in 68/106 (64%). Alternate partners (X::ALK) included ATIC (N=24) and CTLC, COL1A2, MSN, MYH9, RNF213, SATB1, TFH, TPM3, and TRAF1 (1-3 cases each). X::ALK was associated with older age (mean, 52 y) than NPM1::ALK (32 y; P&lt;0.0001) and showed relative overexpression of 49 genes, including multiple activators of small GTPases such as CGNL1 (FC, 5.6; Padj=2.4×10-6), SRGAP1, ALS2, DOCK1, and NCKAP1. ALK expression was similar in cases with NPM1::ALK and X::ALK and there was no significant difference in OS. Non-ALK TK fusions were seen in 17/273 ALK− cases (6%), including TYK2 (N=9); JAK2 (N=6); and ROS1 (N=2). All were Type I ALCLs (P&lt;0.0001). ALK− cases with non-ALK TK fusions overexpressed 38 genes compared to ALK− cases without TK fusions, including cytokines involved in the IL17 signaling pathway such as CXCL6 (FC, 12.5; Padj=4.2×10-3), CXCL1, and CSF3. We then assessed the relationship between ALK− cases with non-ALK TK fusions and ALK+ cases by separately comparing each subset to ALK− cases without TK fusions. FC values for ALK− cases with non-ALK TK fusions were significantly correlated with FC values for ALK+ ALCLs over the set of expressed genes (R=0.54; P&lt;0.0001). At a median follow-up of 32 months, 0/4 systemic ALCL patients with non-ALK TK fusions had died, while 30% of those with ALK− ALCL without TK fusions had died, but this was not statistically significant (P=0.26). The other 13 ALK− cases with non-ALK TK fusions were either localized ALCLs or did not have outcome data available. Of 20 TP63 fusions, 17 (85%) were TBL1XR1::TP63 and 3 were X::TP63. As previously reported, TP63 fusions were associated with poor OS (median OS, 10 months; not reached for other ALK− ALCLs; P=0.007). Four ALCLs had a novel NCL::UBTF fusion involving genes encoding the nucleolar proteins nucleolin and nucleolar (upstream binding) transcription factor-1. Four cases each had PARG::BMS1 and TNK1::GPS2 (the latter seen only in Type I ALCLs). Conclusions: This large consortium-based analysis of coding fusions further elucidates the molecular distinction between Type I and Type II ALCLs. Both ALK and non-ALK TK fusions were seen exclusively in Type I ALCLs, which we have shown previously are enriched for JAK-STAT3 pathway genes and correlate strongly with pSTAT3Y705 positivity by immunohistochemistry. These fusions were not identified in Type II ALCLs, which previous data suggest are associated with epigenetic alterations. ALK+ ALCLs with NPM1::ALK and X::ALK showed differences in gene expression, suggesting biological differences. Furthermore, ALCLs with non-ALK TK fusions shared gene expression features with ALK+ ALCL. Clinical fusion detection could enhance molecular classification of Type I and Type II ALCLs and may guide precision therapy.

Introduction Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of clinically aggressive entities. ~30% of cases cannot be further classified and are designated as PTCL - not otherwise specified (NOS). We delineated two novel molecular subgroups among PTCL-NOS with distinct clinical and biological features: PTCL-GATA3 which is characterized by high expression of GATA3, a master regulator of T-helper-2-cell (TH2) differentiation, and its target genes (i.e., CCR4, IL4, IL13) and PTCL-TBX21 which is characterized by high expression of TBX21, a master regulator of TH1 differentiation, and its target genes (i.e., CXCR3, IFNG), with the GATA3 subtype showing significantly worse clinical outcomes (overall survival). Nonsense mutations in CCR4 are found predominately in PTCL-GATA3, often within the cytoplasmic domain and gain-of-function in nature. Methods Mutation and copy number data were generated from whole exome sequencing (TFH phenotype excluded following LLMPP pathology review; n = 153). Single nucleotide variants were determined using Mutect2 and Varsvan2 using in-house filtering. Copy number (CN) profiles were generated using CNVkit. Four CCR4 cytoplasmic domain mutants and controls (cytoplasmic domain knockout [CDKO], wild-type CCR4 overexpression (CCR4-WT), and empty vector [EV]) were transduced into healthy-donor CD4+ T cells (n = 2). Polarization studies were generated using TH1- or TH2-associated cytokine exposure. Surface plasmon resonance was used to determine the binding affinity of CCR4 proteins to CCL17 and CCL22. In silico docking studies were done through Schrödinger's BioLuminate. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was performed through co-incubation of NK92 (CD16 and CD56) and CCR4 edited primary CD4+ T-cells (stained with calcein AM) for 2 hours at a 5:1 NK:T cell ratio by 7-AAD flow cytometry. Results PTCL-GATA3 had a higher frequency of CN aberrations (e.g. TP53, PTEN CN loss) and a more aberrant genome (p &lt; 0.001) than PTCL-TBX21, including mutations with genes associated with DNA repair and damage (TP53, ATM) and T-cell differentiation (CCR4). CCR4 mutations were virtually mutually exclusive with TP53 mutations, and these cases showed higher CCR4 mRNA and protein expression than CCR4-WT cases (p &lt; 0.05). Primary CD4+ T-cells with ectopic expression of the CCR4 variants, especially CCR4-Q330X, did not require CD3/CD28 for proliferation, bypassing CD3/CD28 ligation for T-cell activation. In addition, proliferation rates were similar between TH2-conditional media and normal media, but proliferation and TBX21 expression in TH1-conditional media were inhibited, implying a novel role of CCR4 in T-cell receptor (TCR) activation and TH-differentiation. CCR4 variant proteins showed higher binding affinity for their natural ligands, CCL22 and CCL17, with mutants exhibiting tighter binding to both ligands (1.5- to 3-fold decrease in KD) by surface plasmon resonance and in silico modeling studies. The in silico models demonstrated mutant CCR4 bound mogamulizumab tighter than CCR4-WT, consistent with a higher ADCC-induced apoptosis rate, highest with CCR4-Q330X mutation. CCR4-Y331X exhibited a worse docking score compared to CCR4-WT and a lower ADCC-induced apoptosis rate. Conclusions Our findings reveal distinct genetic characterizations including aberrant TP53 and PI3K signaling and CCR4 mutations in PTCL-GATA3. These CCR4 mutations are shown to have a key role in PTCL-GATA3 pathogenesis by dysregulating TH-differentiation and aberrantly activating the TCR. CCR4 mutation promotes the TH2 phenotype by blocking TH1 differentiation. CCR4 mutant proteins demonstrated an increased binding affinity towards its known ligands, CCL17 and CCL22, and mogamulizumab, consistent with previous studies exhibiting mogamulizumab efficacy against CCR4 mutations. However, the most frequent CCR4 mutant, Y331X, showed worse binding compared to other CCR4 mutants or CCR4-WT, a mutation that showed Mogamulizumab-resistance in a prior CTCL study as well as in an in vitro ADCC assay. The results emphasize aberrant pathways implicated by genomic and clinical evaluation of two novel PTCL subtypes, as well as support the novel role of mutant CCR4 in enhancing T-cell lymphomagenesis and T-helper differentiation, highlighting multiple therapeutic targets.

Introduction Plasmablastic lymphoma (PBL) is a rare aggressive subtype of lymphoma. It is more common in HIV positive patients, and is often associated with Epstein-Barr virus (EBV) and MYC rearrangements. PBL is characterized by markers of plasmacytic differentiation including CD138 and MUM1/IRF4, and loss of B-cell markers such as CD20, which suggests a cell of origin similar to that of multiple myeloma (MM) or activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL). The standard of care for PBL is CHOP-based chemotherapy but this is frequently not curative. Other than EBV-negative status, which defines a high-risk group, no other biological features have been discovered that are associated with outcomes. Methods Here, we provide a comprehensive analysis of the genomic and transcriptomic landscape of PBL using a collection of 176 exomes and genomes (n=55 new, contributed by Lymphoma/Leukemia Molecular Profiling Project [LLMPP] sites and centrally reviewed by the LLMPP pathology panel [WHO 2016]; n=121 previously published [PMID: 33225311, 33951889, 34465776, 34714908]), 65 transcriptomes (n=44 new; n=21 published) and 37 in situ single cell transcriptomic profiles (CosMx Spatial Molecular Imager) from archival diagnostic tissue biopsies. Somatic mutations (SNVs/Indels) were identified using an ensemble of four variant callers (Strelka2, Lofreq, Mutect2, SAGE). Salmon and DESeq2 were used to identify differentially expressed genes. This multi-omics dataset was compared to other known B-cell malignancies including 92 MM, 238 Burkitt lymphomas (BL), and 208 DLBCLs. Cell segmentation of the CosMx data was performed using Baysor and downstream analyses were conducted using the Seurat workflow. For functional validation, cell viability assays (CellTiter-Glo Luminescent Assay) were conducted in the PBL cell line, PBL-1, and 4 control DLBCL cell lines. Results Within the cohort, 63% (106/169) of primary PBL cases were EBV-positive (EBV+) and 57% (82/144) harbored MYC rearrangements (MYC+). When stratifying patients by EBV and MYC rearrangement status, mutations in different genes were enriched in each group: STAT3 occurred in 55% (29/53) of EBV+/MYC+ PBLs; NOTCH1 in 19% (7/36) of EBV+/MYC-; MYC in 24% (6/25) and TP53 in 40% (10/25) of EBV-/MYC+ tumors; TET2 was mutated in 38% (9/24) and KRAS in 25% (6/24) of EBV-/MYC- PBLs. EBV-/MYC+ tumors represented a subgroup of patients with dismal outcomes. The 2-year overall survival (OS) of these patients was 22% compared to 61%, 78%, and 54% of EBV+/MYC+, EBV+/MYC-, and EBV-/MYC- PBL patients, respectively (log-rank test p-adj. &lt; 0.05). Consistent with previous studies, we observed recurrent mutations affecting JAK-STAT (STAT3, SOCS1, SOCS3, DUSP2) in 41% of cases and RAS-RAF (NRAS, KRAS, BRAF) signaling in 38% of all PBLs. In contrast to ABC-DLBCL, very few mutations occurred in the NF-κB signalling pathway. Accordingly, PBL showed marked downregulation of genes involved in B-cell receptor and NF-κB signaling. Consistent with the lack of reliance on these pathways, functional analyses in PBL-1 showed resistance to treatment to ibrutinib as well as PI3K- and MALT1-inhibitors. Moreover, constitutive expression of the IκBα super-repressor did not affect viability of PBL-1 cells. In situ single cell transcriptomic data analyses showed a sparse tumor microenvironment (TME) in PBL, with the primary non-malignant cell types being, on average, 10% macrophages, 7% NK/T cells, and 4% stromal cells of all represented cell types. EBV+/MYC+ tumors displayed a more abundant immune cell population with higher levels of macrophages (median 12%) and NK/T cells (median 9%), whereas EBV-/MYC- tumors were composed of the lowest levels of immune cells (median 4% macrophages and 1% NK/T cells). With respect to malignant cell phenotypes, we noted a novel population of SPP1-expressing cells that were enriched in EBV+/MYC+ tumors and a population of CD44-expressing cells that were enriched in EBV+/MYC- tumors. Conclusion Here we present novel subgroups of PBL with unique biology and clinical outcomes. We show that PBL does not rely on B-cell receptor signaling, instead relying on potentially targetable vulnerabilities in JAK-STAT, NOTCH, and RAS-RAF signaling according to EBV positivity and MYC rearrangement status. Lastly, we provide insight into the TME of PBL and demonstrate a novel population of SPP1 and CD44 expressing malignant cells in EBV+ tumors.

Abstract Recent introduction of 2 different lymphoma classifications has raised concerns about consistency in diagnosis, management, and clinical trial enrollment. Data from a large cohort reflecting real-world clinical practice suggest that differences between the classifications will affect &lt;1% of non-Hodgkin lymphomas.