Supplementary Table S1 - Differentially expressed genes and PAGE analysis in Myd88 L252P GCB

Gating strategies, validation of results using alternative models or definitions, QC controls, etc.

Journal Article Reply to “Kikuchi disease and COVID-19 vaccination” Get access Jeffrey W Craig, MD, PhD, Jeffrey W Craig, MD, PhD University of Virginia Health System, Charlottesville, VA, US jeffrey.william.craig@gmail.com https://orcid.org/0000-0003-1295-3258 Search for other works by this author on: Oxford Academic Google Scholar Pedro Farinha, MD, PhD, Pedro Farinha, MD, PhD BC Cancer and University of British Columbia, Vancouver, Canada Search for other works by this author on: Oxford Academic Google Scholar Aixiang Jiang, MS, Aixiang Jiang, MS BC Cancer and University of British Columbia, Vancouver, Canada Search for other works by this author on: Oxford Academic Google Scholar Brian Skinnider, MD, Brian Skinnider, MD BC Cancer and University of British Columbia, Vancouver, Canada Search for other works by this author on: Oxford Academic Google Scholar Graham W Slack, MD, Graham W Slack, MD BC Cancer and University of British Columbia, Vancouver, Canada Search for other works by this author on: Oxford Academic Google Scholar Andrew Lytle, MD, PhD Andrew Lytle, MD, PhD BC Cancer, Vancouver, Canada Search for other works by this author on: Oxford Academic Google Scholar American Journal of Clinical Pathology, aqad118, https://doi.org/10.1093/ajcp/aqad118 Published: 12 October 2023

Introduction: Classic Hodgkin lymphoma (CHL) is currently classified into subtypes based on morphology and pathological features. However, there is accumulating evidence that subtypes of CHL can also be distinguished by their genomic profiles. To delineate molecular subtypes of CHL, we applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events (Aoki, et al. ASH 2023). In particular, we found frequent CSF2RB mutations in cluster 1, which is characterized by mutations in TNFAIP3, CSF2RB and GNA13. CSF2RB is the common signaling subunit of the cytokine receptors for IL-3, IL-5, and GM-CSF, with cytokine-independent activating mutations previously reported in the transmembrane and extracellular domains in pediatric T-cell acute lymphoblastic leukemia, and breast cancer, respectively. However, the functional role of CSF2RB mutations in CHL pathogenesis remains unknown. Materials and methods: We performed targeted and whole exome sequencing of Hodgkin and Reed-Sternberg (HRS) cells (n=116) purified using laser capture microdissection and flow cytometry (FCM)-based cell sorting. For in vitro functional studies, we retrovirally expressed CSF2RB WT or mutants observed in CHL tumors into two CHL-derived cell lines (L428, U-HO1). Using these isogenic models, we characterized the full spectrum of domain-specific mutations and elucidated the role of CSF2RB alterations in oncogenic signaling. Moreover, to investigate spatial tumor microenvironment architecture, we performed imaging mass cytometry (IMC) on the exome/targeted sequencing cohort using a customized antibody panel. Results: In 26/107 cases (24.3%), we identified recurrent CSF2RB mutations, which clustered in the cytoplasmic domain (exon 14). Mutations were categorized as missense (n=2), multiple-hit (n=3), and truncating (n=21) mutations. Functional characterization in the L428 model showed that STAT5 was hyperphosphorylated in cells expressing all truncating mutants observed in cases with CHL (p.S723*, p.E788*, p.Lys838*, p.Q853*, p.Q867*, and p.Gln885*) compared to WT cells in an IL-5-dependent manner. Focusing on the functional consequences of the most commonly observed hotspot, the CSF2RB-p.E788* frameshift mutation, we generated cells with a CSF2RB heterozygous genotype by 1:1 transfection of WT and mutant alleles (WT/p.E788*) in U-HO1. With IL-3 or IL-5 (but not GM-CSF) stimulation, we validated that pSTAT5 was markedly higher in WT/p.E788* compared to WT cells. Differential gene expression analysis by RNA-seq revealed up-regulation of common IL-2 signaling pathway molecules and cytokine gene expression signatures, including genes such as IL-13 and CCL17 (TARC) in p.E788* cells compared to WT cells in the L428 model. These findings were validated in the U-HO1 model with IL-3 or IL-5 stimulation. In accord with the gene expression results, we detected higher amounts of IL-13 and TARC by ELISA in the supernatant from cells expressing all truncating mutants compared with WT cells in the L428 model. Furthermore, these gain-of-function phenotypes could be reversed by a selective JAK2 inhibitor (pacritinib), suggesting deregulated cytokine-dependent JAK2-STAT5 signaling leads to increases in IL-13 and CCL17 protein expression. Next, we performed an in-vitro transwell T-cell migration assay using peripheral blood mononuclear cells from healthy donors and found that supernatant from p.E788* cells significantly enhanced migration of CCR4+ T cells, which contains FOXP3+ Tregs, compared with supernatant from WT cells. Enhanced migration was completely blocked by an anti-CCR4 antagonist. Finally, spatial analysis using IMC data confirmed the close proximity between CCL17+ HRS cells and CCR4+ T cells in primary CHL samples. In particular, we observed a significant enrichment of FOXP3+ Tregs in close proximity to HRS cells in the CSF2RB-mutation enriched molecular cluster. Conclusion: CSF2RB mutations contribute to deregulated, cytokine-dependent, oncogenic signaling, leading to downstream IL-13 related cell-autonomous phenotypes and TARC related tumor microenvironment composition and functions. We predict these phenotypes will pinpoint therapeutically targetable vulnerabilities, and may provide precision therapeutic approaches based on genetic subtype definitions in CHL.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3 , a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Histiocytic sarcoma (HS) is a rare and aggressive hematologic neoplasm characterized by the proliferation of malignant histiocytes. It infrequently presents with periorbital involvement. Here we present the first documented case of ocular adnexal histiocytic sarcoma composite with chronic lymphocytic leukemia/small lymphocytic lymphoma and provide compelling evidence for the transdifferentiation of chronic lymphocytic leukemia/small lymphocytic lymphoma to histiocytic sarcoma in an 80-year-old woman. Comprehending the clinicopathological characteristics of histiocytic sarcoma and various other histiocytic proliferations and neoplasms affecting orbital and ocular structures is imperative for ophthalmic surgeons and pathologists.

MOTIVATION:Single cell segmentation is critical in the processing of spatial omics data to accurately perform cell type identification and analyze spatial expression patterns. Segmentation methods often rely on semi-supervised annotation or labeled training data which are highly dependent on user expertise. To ensure the quality of segmentation, current evaluation strategies quantify accuracy by assessing cellular masks or through iterative inspection by pathologists. While these strategies each address either the statistical or biological aspects of segmentation, there lacks a unified approach to evaluating segmentation accuracy. RESULTS:In this article, we present ESQmodel, a Bayesian probabilistic method to evaluate single cell segmentation using expression data. By using the extracted cellular data from segmentation and a prior belief of cellular composition as input, ESQmodel computes per cell entropy to assess segmentation quality by how consistent cellular expression profiles match with cell type expectations. AVAILABILITY AND IMPLEMENTATION:Source code is available on Github at: https://github.com/Roth-Lab/ESQmodel.

Introduction: Interferon Regulatory Factor 4 (IRF4) is a transcription factor that plays a critical role in the regulation of pre-B-cell development, the germinal center reaction as well as plasma cell differentiation. IRF4 has emerged as a master regulator of cancer-specific gene expression programs in various lymphoid malignancies. Prior studies uncovered a hotspot missense mutation in the DNA-binding domain of IRF4 (C99R) that alters the affinity of binding to specific DNA motifs and leads to disease-specific changes in classic Hodgkin lymphoma (Schleussner et al, 2023). The C99R mutations were also found to be recurrently detected in primary mediastinal large B-cell lymphoma (PMBCL) (Mottok et al, 2019, Noerenberg et al, 2023); however, its functional role in PMBCL pathogenesis remains unknown. Materials and methods: We introduced the C99R mutation into two PMBCL-derived cell lines (U2940, Karpas-1106P) by applying CRISPR prime editing. We then performed ATAC-seq, ChIP-seq, and RNA-seq analyses on our isogenic model systems (IRF4WT and IRF4C99R). We also analyzed single cell (sc) transcriptome data of nuclei isolated from formalin-fixed paraffin-embedded tissues samples of primary PMBCL (n=4) with/without this mutation, including control tissues from reactive lymph node (n=2), and normal thymus (n=2). Results: A meta-analysis of publicly available sequencing data revealed IRF4C99R hotspot mutations in 29 out of 413 PMBCL samples (7%), compared to only 2 out of 1,999 diffuse large B-cell lymphoma samples (0.1%) (p<0.00001). To characterize the role of this mutation, we compared the global chromatin accessibility profiles between IRF4WT and IRF4C99R PMBCL cells by ATAC-seq and identified regions with commonly enhanced (1,334 regions) and reduced (2,796 regions) accessibility across the isogenic models. Specifically, in IRF4C99R cells, the accessibility of flanking regions at specific EICE- and canonical/non-canonical AICE-motifs were elevated but clearly reduced at ISRE motifs. The direct IRF4 binding to these distinct motifs was validated by IRF4 ChIP-seq. In line with these results, differential gene expression analysis revealed down-regulation of key regulators of plasma cell differentiation such as PRDM1 and interferon gamma (IFNγ) signaling-related genes such as TNIK in IRF4C99R cells, both genes were regulated by IRF4 at ISRE DNA motifs. Conversely, a key cytokine in PMBCL CCL17 (TARC) was upregulated at both the mRNA and protein levels in IRF4C99R cells, as validated by qPCR and ELISA, respectively. Previous studies reported downregulation of TARC by IFNγ signaling, a finding that we reproduced by IFNγ stimulation experiments in our PMBCL models. Moreover, we showed that inhibition of TNIK abrogated IFNγ-mediated downregulation of TARC, demonstrating a mechanistic link between IRF4C99R and increased TARC expression. Finally, we found the receptor tyrosine kinase encoding gene EPHB1, regulated at both EICE and AICE motifs, was the highest upregulated gene in IRF4C99R cells (adj. p<0.01). Of note, the higher expression of both TARC and EPHB1 mRNA in primary PMBCL tumors carrying the IRF4C99R (n=3) compared to IRF4WT (n=63) were validated by Illumina Whole-Genome DASL assay. We next performed in vitro binding assays and found EPHB1 specifically interacts with the membrane-bound ligand Ephrin-B2. Functional studies showed Ephrin B2-mediated adhesion and migration were significantly increased in IRF4C99R cells. Moreover, we retrovirally engineered Eu-Myc mouse lymphoma cells to overexpress EPHB1 in an in-vivo syngeneic model. After intraperitoneal injection into C57BL6 mice, the cells with EPHB1 overexpression promoted enhanced tumor growth with increased infiltration of spleen, liver and thymus. Finally, sc transcriptome analysis showed significantly higher EPHB1 expression in primary PMBCL tumor cells with the IRF4C99R mutation (n=2, p<0.001) as compared to cases with IRF4WT (n=2), and the expression was rarely detected in normal B cells. Ephrin-B2 ligand expression was detectable in the endothelial cells of PMBCL tumors. Conclusion: Our findings highlight pleiotropic effects of the heterozygous IRF4C99R mutation leading to hallmarks of PMBCL lymphomagenesis including a plasmacytic differentiation block, upregulation of the key cytokine TARC, and providing a potential explanation of disease specific tropism through the Ephrin-B2/EPHB1 axis.

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

Introduction: Diffuse large B-cell lymphomas (DLBCLs) with a non-germinal center B cell-like (non-GCB) cell-of-origin are frequently driven by genetic alterations that culminate in constitutive B-cell receptor (BCR) signaling, which has inspired the exploration of Bruton's tyrosine kinase inhibitors (BTKi) in these lymphomas. However, the phase III PHOENIX study that randomized untreated, non-GCB DLBCL patients to R-CHOP plus placebo or ibrutinib failed to meet its primary endpoint of event-free survival (Younes et al. 2019), which suggests that cell-of-origin alone is an insufficient biomarker to predict BTKi sensitivity in DLBCL. More recently, a DLBCL genetic classifier termed LymphGen has identified distinct subtypes (MCD and N1) of non-GCB DLBCL that benefit from the addition of BTKi to R-CHOP (Wilson et al. 2021). However, genetic classifiers are complex and difficult to implement in routine clinical settings and may fail to capture all DLBCLs that benefit from BTKi. Therefore, we sought to identify a straightforward biomarker of BTKi responsiveness in DLBCL with greater precision than cell-of-origin but with broader inclusivity than current genomic platforms, such as LymphGen. We hypothesized that CD5 - a surrogate marker of BCR activation - may effectively identify BCR-driven, non-GCB DLBCLs that are sensitive to BTKi therapy, and evaluated the extent to which CD5 protein expression and a transcriptionally defined CD5 gene signature could accurately identify BCR-activated DLBCLs with potential susceptibility to BTKi-based therapies. Methods: CD5 immunohistochemistry (IHC) was performed on a cohort of 406 diagnostic DLBCL samples, which were considered CD5+ if >=30% of lymphoma cells exhibited unequivocal membranous staining. A majority of DLBCL samples had available RNA-sequencing and targeted mutational sequencing data. A comparison of differentially expressed genes between CD5+ and CD5- DLBCLs was performed in order to construct a 60-gene CD5 signature (CD5sig), which was applied to large genomic DLBCL datasets, including pre-treatment biopsies from patients enrolled on PHOENIX (n = 584) to evaluate the utility of the CD5sig in identifying DLBCLs that benefitted from the addition of ibrutinib to R-CHOP. Results: Twenty-six of 406 DLBCLs were identified as CD5+ by IHC (6% of all DLBCLs; 12% of non-GCB DLBCLs). CD5 IHC+ DLBCLs were majority non-GCB cell-of-origin and were associated with inferior progression-free survival (PFS) to R-CHOP (50% 3-year PFS), compared with CD5 IHC- DLBCLs, consistent with previous reports. Gene set enrichment analysis revealed that CD5 IHC+ DLBCLs exhibited transcriptional features of BCR activation, and mutational analysis demonstrated that CD5 IHC+ DLBCLs were enriched for CD79B BCR-activating mutations known to correlate with BTKi sensitivity. Many CD5 IHC+ DLBCLs, however, lacked canonical BCR-activating mutations or were classified as “Other” by LymphGen. A CD5 gene signature (CD5sig; Figure 1A) was developed that recapitulated these findings in independent DLBCL datasets (NCI, Duke), where ~13% of non-GCB DLBCLs were classified as CD5sig+. Together, these results suggest that CD5 signature expression captures DLBCLs with both a genetic and non-genetic basis for BCR dependence. Supporting this notion, CD5sig+ DLBCL patients (< 60 years) derived a selective and striking event-free and overall survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study ( Figure 1B), independent of LymphGen classification. Conclusions: We demonstrate that CD5 IHC and a novel CD5 gene signature identify high-risk, BCR-driven DLBCLs. Importantly, the CD5 signature also identifies DLBCL patients with a selective survival advantage to BTK inhibitor-based therapy, independent of LymphGen classification. In conclusion, the CD5 signature expands upon LymphGen classification as a biomarker of BTKi response by accurately identifying DLBCLs with both genetic and non-genetic bases for BTKi response. The CD5 signature and/or CD5 IHC should be prospectively evaluated in BTKi-based clinical trials for non-GCB DLBCLs.

Introduction: Gene expression profiling has identified DLBCLs that bear transcriptional similarity to either non-malignant germinal center B-cells (GCB-DLBCL) or activated B-cells (ABC-DLBCL, i.e. non-GCB-DLBCL). This cell-of-origin (COO) classification has guided precision medicine strategies, including the use of ibrutinib, a BTK inhibitor (BTKi), with ABC-DLBCLs thought to be sensitive to BTKi due to their constitutive activation of NF-kB downstream of the B-cell receptor (BCR). However, the phase III Phoenix trial testing the addition of ibrutinib to R-CHOP chemoimmunotherapy surprisingly failed to demonstrate a survival benefit in non-GCB-DLBCLs. By employing a newly defined genetic classification algorithm (LymphGen), distinct genetic DLBCL subtypes that benefit from this regimen were identified. However, LymphGen is not utilized in the clinic and fails to classify >40% of DLBCLs. To overcome these challenges, we sought to identify a straightforward biomarker of BTKi sensitivity. CD5 was selected as a potential candidate, given its role as a negative regulator of BCR signaling and expression in other BTKi-responsive B-cell lymphomas. Methods: CD5 IHC was performed on a cohort of 405 DLBCLs, most with paired RNA and targeted mutational sequencing. Comparison of the transcriptomes of CD5+ and CD5− DLBCLs was used to construct a 60-gene CD5 signature (CD5sig), which was applied to large genomic DLBCL datasets, including pre-treatment biopsies from the Phoenix trial (n = 584) to evaluate its utility in predicting improved response to R-CHOP + ibrutinib. Results: Twenty-seven DLBCLs were identified as CD5+ by IHC (6% of cases; 12% of non-GCB). Consistent with previous reports, CD5+ DLBCLs were enriched for a non-GCB COO and exhibited inferior progression-free survival to R-CHOP. CD5+ DLBCLs upregulated genes related to BCR signaling and had a significantly higher incidence of CD79B BCR-activating mutations. A CD5 signature (CD5sig) was created from differentially expressed genes between CD5 IHC+ and CD5 IHC− non-GCB-DLBCLs. When applied to an external dataset of 349 non-GCB-DLBCLs, CD5sig+ DLBCLs (15% of cases) were enriched for BCR-activating mutations and were majority MCD (MYD88 and CD79B) or unclassified by LymphGen. CD5sig+ DLBCLs were largely devoid of BCL10 activating mutations known to drive BTKi resistance. When applied to the Phoenix study, CD5sig+ DLBCLs selectively benefitted from R-CHOP + ibrutinib by event-free and overall survival. This survival difference was preserved even among LymphGen-unclassified DLBCLs. Conclusions: Here, we identify CD5 expression as a simple and useful biomarker to identify high-risk, BCR-driven DLBCLs beyond those identified through genetic classification. Based upon unique transcriptional features of CD5 IHC+ DLBCLs, we developed a novel CD5 signature that identifies DLBCLs vulnerable to BTKi therapy. Citation Format: Alan Cooper, Sravya Tumuluru, Kyle Kissick, Girish Venkataraman, Nikita Kotlov, Aleksander Bagaev, Andrew Lytle, Gerben Duns, David W. Scott, Christian Steidl, Brendan Hodkinson, Srimathi Srinivasan, Justin Kline, James Godfrey. A CD5 signature identifies diffuse large B-cell lymphomas (DLBCLs) sensitive to Bruton’s tyrosine kinase (BTK) inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4330.

Supplementary Data from BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL

Objectives: Multiple case reports describe Kikuchi-Fujimoto disease (KFD) following COVID-19 vaccination, but the true nature of this phenomenon is unknown. The purpose of this study was to further assess the relationship between KFD and COVID-19 vaccination at the population level.Methods: Confirmed KFD cases from January 2018 to April 2022 were identified from provincial pathology archives and analyzed in the context of vaccination statistics from public health resources.Results: Our statistical models provide evidence of a temporal association between KFD and both antecedent COVID-19 vaccine administration as well as age-stratified vaccination rates. Eight new cases of plausible COVID-19 vaccine-associated KFD are presented, collectively exhibiting clinical and pathologic features that overlap substantially with those of idiopathic KFD.Conclusions: Our findings indicate that KFD is observed in association with COVID-19 vaccination and suggest that mechanistic studies are warranted.

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

Abstract A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients. Significance: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8–11). This article is highlighted in the In This Issue feature, p. 1

CD19 directed CAR T-cell therapy is used to treat relapsed/refractory B-cell acute lymphoblastic leukemia. The role of the pre-CAR bone marrow (BM) stromal microenvironment in determining response to CAR T-cell therapy has been understudied. We performed whole transcriptome analysis, reticulin fibrosis assessment and CD3 T-cell infiltration on BM core biopsies from pre- and post-CAR timepoints for 61 patients, as well as on a cohort of 54 primary B-ALL samples. Pathways of fibrosis, extracellular matrix development, and associated transcription factors AP1 and TGF-β3, were enriched and upregulated in nonresponders (NR) even prior to CAR T cell therapy. NR showed significantly higher levels of BM fibrosis compared to complete responders by both clinical reticulin assessment and AI-assisted digital image scoring. CD3+ T cells showed a trend toward lower infiltration in NR. NR had significantly higher levels of pre-CAR fibrosis compared to primary B-ALL. High levels of fibrosis were associated with lower overall survival after CAR T-cell therapy. In conclusion, BM fibrosis is a novel mechanism mediating nonresponse to CD19-directed CAR T-cell therapy in B-ALL. A widely used clinically assay for quantitating myelofibrosis can be repurposed to determine patients at high risk of non-response. Genes and pathways associated with BM fibrosis are a potential target to improve response.

Abstract Activated B cell–like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11–BCL10–MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. Significance: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure–function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825

Introduction CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) remains the standard treatment for older adults with non-anaplastic large cell lymphoma (ALCL), nodal peripheral T-cell lymphomas (PTCLs). However, recent studies suggest that patients (pts) > 75 years (y) have similar outcomes with CHOP compared to those receiving palliative regimens (Ellin et al. Hematological Oncology 2017). In addition, with studies demonstrating sensitivity of PTCLs to epigenetic therapies, especially in the T-follicular helper (TFH) subtypes, there are emerging clinical trials evaluating these agents in newly diagnosed PTCL patients > 60 y (NCT02232516). We evaluated the long-term outcomes of pts aged 65 y or older with PTCLs, a group generally not transplant eligible, treated with systemic therapy with a focus on those treated with curative intent CHOP(like) chemotherapy. Methods The BC Cancer Lymphoid Cancer Database was reviewed and pts > 65 years with PTCL, not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL) as well as other T-Follicular Helper PTCL (collectively referred to as TFH T-cell lymphoma [TFHTCL]) diagnosed between 2000-2021 and who received front-line chemotherapy were included. Diagnoses were based on the World Health Organization (WHO) classifications of 2001, 2008, and 2017, depending on the era of diagnosis. Time to progression (TTP) was measured from the date of pathologic diagnosis to the date of relapse/progression, death due to treatment toxicity or lymphoma. Results 152 pts were initially identified, of which 5 were excluded: stage IA/IAE (n=3); CNS involvement only (n=2). Of the remaining 147 pts, 67 (46%) had PTCL-NOS and 80 (54%) had TFHTCL. The median age was 74 y (range 65-90) and the majority of pts had high risk features: International Prognostic Index (IPI) score of > 3 (n=113, 76%), stage III/IV disease (n=136, 93%) and PS of > 2 (n=85, 58%). Most pts (n=127, 86%) received multi-agent chemotherapy with curative intent. All pts received CHOP/CHOP-like chemotherapy (including five pts treated with CHP-brentuximab vedotin)Over half of these patients (n=66, 52%) received attenuated doses of chemotherapy starting from cycle 1, typically 50-75% dose intensity. Only two patients received consolidative autologous stem cell transplant. The remaining 20 patients received chemotherapy with palliative treatment intent, the majority (n=18, 90%) receiving cyclophosphamide alone or with the CVP (cyclophosphamide, vincristine, and prednisone) regimen. The median follow-up of all pts using the reverse Kaplan-Meier method was 9.2 y (range 0.9 to 12.5). The estimated 5-y TTP and overall survival (OS) for all patients were 21%, and 26%, respectively. Outcomes were similar in PTCL-NOS and TFHTCL (5-y TTP 17% vs 25%; p = 0.26). Curative intent chemotherapy was used more frequently in the 65-74 y vs > 75 y age group (70% vs 36%, p = 0.004). As expected, outcomes were superior in those treated with curative intent (Table 1): Median TTP of 11.3 months (m) vs 3.0 m; median OS of 24 m vs 4.5 m. Improvements in TTP and OS extended to those > 75 y (p < 0.001 for both). Considering the curative intent cohort, 91% of the relapse/progression events occurred within the first 3 y, and there was no significant difference in outcomes (5-y TTP/OS) with increasing age: 65-75 (19%/25%), >75 (34%/35%) (p = 0.08/0.6). Further, the outcomes of pts who received attenuated doses of CHOP-like chemotherapy were not inferior compared to full dose chemotherapy (5-y TTP/OS 32%/37% vs. 17%/21% p= 0.2/0.3). 5-y TTP/OS by the IPI ranged from 30%/52% for IPI of 1-2 to 12%/14% for IPI 4-5. The prognostic index for T-cell lymphoma (PIT) was more effective at identifying a lower risk group: 5-y TTP/ OS (PIT 1 40%/57%; PIT 2 21%/28%; PIT 3 21%/20% PIT 4 9%/10%, p = 0.002/0.001). Both models were prognostic in PTCL-NOS and TFHTCL. (Table 1) Conclusion With mature follow-up, ~ 30% of all older pts treated with curative intent CHOP-like chemotherapy remain progression-free and are alive at 5 y. Although results remain suboptimal, robust older patients can still be considered for curative intent CHOP chemotherapy with tailored doses to mitigate toxicity. Those with low risk IPI/PIT scores have a more favourable outcome with this approach, with a 5-y OS of > 50%, suggesting a benefit of subsequent therapies. In contrast, higher risk disease have poor outcomes and novel therapeutic approaches should be considered. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and the activated B cell-like subtype (ABC-DLBCL) is the most aggressive form and harbors frequent mutations of immune signaling pathways that culminate in constitutive activation of CARD11-MALT1-BCL10 (CBM) complex and downstream NF-κB pathway. CBM complexes form large macromolecular structures due to signal-induced polymerization of the BCL10 subunit, which is affected by recurrent somatic mutations in ABC-DLBCLs. Through biochemical, structural and functional dissection of these mutations, we find that BCL10 mutations fall into two functionally distinct classes: missense mutations of the BCL10 CARD domain (hotspot R58Q) and truncation of its C-terminal tail (hotspot E140X). To explore the functional consequences of BCL10 mutations, we established reporter systems to evaluate their impact on MALT1 and NF-𝜅B activities which are BCL10 downstream signaling cascades. We found that almost all mutants induced aberrantly strong NF-𝜅B and MALT1 activities in lymphoma cells as compared to WT BCL10, indicating the gain-of-function effect of BCL10 mutations. By performing immunohistochemistry staining of p65 in a set of tumor tissue microarray from DLBCL patients (n=298), we revealed that BCL10 mutant tumors have significantly (Mann-Whitney p<0.0001) increased p65 nuclear staining score compared to BCL10 WT tumors, suggesting enhanced NF-𝜅B activity. To investigate the biochemical impact of BCL10 mutants on CBM complex formation, we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 proteins and found that both BCL10R58Q and BCL10E140X manifested faster and more spontaneous polarization compared to BCL10WT. Surprisingly, through mapping the BCL10-MALT1 interaction, we found that truncating mutation (E140X) abrogated a novel protein interaction motif through which MALT1 inhibits BCL10 polymerization, thus unleashing spontaneous CBM filament formation and inducing addiction to MALT1 activity. In marked contrast, the CARD missense mutation (R58Q) on BCL10 filament interface not only does not disrupt but enhances filament formation and it also alters CBM complex kinetics forming glutamine network structures that stabilize BCL10 filaments, but this still may require the upstream signal to activate MALT1. Importantly, we found that BCL10 mutant cells were less dependent on upstream CARD11 activation in MALT1 activation, NF-𝜅B signaling and cell growth assays performed in ABC-DLBCL lines. Furthermore, in vitro and in vivo xenograft studies revealed that BCL10 mutant lymphomas are resistant to BTK inhibitors, whereas BCL10 truncating (E140X) but not missense CARD (R58Q) mutants were hypersensitive to MALT1 protease inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL and further precision can be achieved by tailoring therapy (e.g. MALT1 inhibitors that are currently being tested in clinical trials) according to specific biochemical effects of distinct mutation classes. Citation Format: Min Xia, Liron David, Matthew R Teater, Johana Gutierrez, Xiang Wang, Cem Meydan, Andrew Lytle, Graham Slack, David Scott, Ozlem Onder, Kojo Elenitoba-Johnson, Nahuel Zamponi, Leandro Cerchietti, Tianbao Lu, Ulrike Philippar, Lorena Fontan, Hao Wu, Ari Melnick. BCL10 mutations define distinct dependencies guiding precision therapy for DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A28.