Germinal Center B-cell Research Articles

A Phosphatase Membrane-Shuttle Enables Feedback Control of B-Cell Signaling

Mechanisms of local kinase recruitment to amplify signaling in lipid rafts have been extensively studied. However, mechanisms of phosphatase membrane-recruitment to terminate local signaling activity remained elusive. Here we identified CD25 (IL2Rα) as a central phosphatase membrane-shuttle and feedback regulator of B-cell receptor (BCR)-signaling in activated and transformed B-cells. This was unexpected because CD25 is known as one of three chains of the IL2-receptor on T- and NK-cells. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells recruits an inhibitory phosphatase complex for feedback control of BCR-signaling and its oncogenic mimics. Reminiscent of genetic phosphatase-ablation, conditional CD25-deletion profoundly depleted early B-cell development followed by dramatic expansion and hyperactivation of mature B-cell subsets and autoimmunity owing to imbalances of BCR-signaling. However, defects in CD25 deficient mice were not replicated in mice that express CD25 but lack expression of the IL2 cytokine, demonstrating IL2-independent functions of CD25 in B-cells. In six clinical cohorts, high expression levels of CD25 predicted poor outcomes for patients with pre-germinal center (GC), in contrast to favorable outcomes for patients with post-GC B-cell malignancies. Consistent with opposite clinical outcome-annotation, genetic CD25-deletion in B-cell precursor leukemia induced rapid cell death but accelerated proliferation in post-GC B-cell lymphomas. Mechanistically, BCR-engagement or oncogenic BCR-signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail at S268. Genetic deletion, in vitro kinase, and interactome studies revealed that PKCδ-dependent CD25-S268 phosphorylation triggered the PKCδ-scaffold RACK1 to recruit inhibitory phosphatases (SHP1, SHIP1) for membrane-translocation and feedback control of BCR-signaling. Deletion of CD25 in human B-cells induced autonomous BCR-signaling and Ca2+-oscillations: In the absence of CD25, inhibitory phosphatases were no longer recruited to the cell membrane to control duration and strength of BCR-signaling. Genetic rescue experiments identified membrane-tethered RACK1 and CD25-S268 tail-phosphorylation as central structural elements of this previously unrecognized phosphatase membrane-shuttle. As a result, CRISPR-mediated knock-in of CD25-S268A mutant that abrogates the formation of inhibitory phosphatase complexes caused accelerated proliferation with hyperactivation of BCR-signaling in primary human tonsillar germinal center B-cells. In conclusion, PKCδ-mediated phosphorylation of CD25 assembles RACK1-dependent inhibitory phosphatase complexes to enable feedback control of BCR-signaling to prevent autoimmunity and malignant transformation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Serum Proteome Profiling Reveals Inflammatory, Molecular and Prognostic Information Beyond the Ctdna in Aggressive B-Cell Lymphomas

Introduction Large B-cell lymphomas (LBCL) constitute a group of aggressive B-cell lymphomas, which have striking genetic heterogeneity, variable tumor microenvironment (TME), clinical presentation and treatment outcome. Although LBCL patients are treated with a curative intent, their survival is suboptimal, and the current prognostic tool, the International Prognostic Index (IPI), cannot capture the biological heterogeneity of the disease. We studied serum proteome in a uniformly treated cohort of the patients with LBCL to characterize host response to lymphoma and to identify clinically relevant circulating proteins. Materials and methods We applied proximity extension assay of 1463 proteins (Olink Proteomics) to pretherapeutic serum samples of 109 patients with primary LBCL and five healthy volunteers. The LBCL patients included in the study were 21-65 years of age and had clinically high-risk disease (age adjusted IPI ≥ 2) and/or site-specific risk factors for central nervous system (CNS) progression. Patients were treated in a Nordic phase II protocol with a dose-dense immunochemotherapy and early CNS prophylaxis. The findings from the serum proteome profiling were correlated with clinical and molecular data (gene expression, n = 48; multiplex immunohistochemistry, n = 39) from tumor tissue and circulating tumor DNA (ctDNA) from concurrent plasma samples (n = 91). Results Unsupervised analysis of the pre-treatment serum protein levels revealed a subgroup of patients (n=24, 22%) with a distinct protein profile compared to the other patients and healthy volunteers (Figure 1A). The patients with this profile had elevated levels of inflammatory proteins, such as interferon gamma (IFNg) related T-cell response markers. Clinically, the patients with the inflamed serum protein profile had more often B-symptoms and lymphoma infiltration in the bone marrow, and dismal survival as compared to the patients with the non-inflamed profile (overall survival, HR 4.31, 95%-CI 1.71-10.88, P < .01, Figure 1B). In addition, T-cell/histiocyte rich large B-cell lymphoma and activated B-cell (ABC) molecular subtype were more common entities in these patients. Lymphomas in the inflamed serum cohort were characterized by a higher proportion of tumor-infiltrating T-lymphocytes and macrophages expressing immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1), T-cell immunoglobulin mucin-3 (TIM3) and lymphocyte-activation gene 3 (LAG3). In liquid biopsy, a striking association between the inflamed serum protein profile and high plasma ctDNA levels was discovered (P < .0001). However, the profile associated with poor outcome independently of ctDNA levels (P < .01). Integrative analysis of ctDNA and serum proteins revealed synergistic potential for tumor profiling that extended beyond inflammatory markers. For example, circulating protein levels of tumor protein 53 (TP53) associated with p53 mutations, TP53 expression in the lymphoma tissue and high tumor burden. Moreover, high levels of serpin family A member 9 (SERPINA9) and TNF receptor superfamily member 13B (TNFRSF13B) characterized germinal center B-cell like/EZB and ABC/MCD diffuse large B-cell lymphomas, respectively. Conclusions Our findings reveal an inflamed serum protein profile, which reflects host response to lymphoma, correlates with TME features, extends beyond ctDNA characteristics, and translates to poor outcome. Moreover, by integrating serum protein and plasma ctDNA profiling we uncover new associations that broaden the opportunities of liquid biopsies in LBCL. Together, our data establish a novel interaction between lymphoma and the host. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Impact on Survival of the Introduction of New Therapies for Relapsed-Refractory Aggressive B-Cell Lymphoma, Based on the Relinf Registry: A Study By the Spanish Geltamo Group

Introduction. Treatment of relapsed-refractory (r/r) diffuse large B-cell lymphoma (DLBCL) and high-grade B-cell lymphoma (HGBCL) has changed substantially in recent years, due to the incorporation (in a commercial setting or in the context of clinical trials) of new therapeutic options such as new monoclonal antibodies (MA), antibody-drug conjugates (ADC), bispecific antibodies (BA) or CAR-T cell therapy. The main objective of our study was to evaluate the frequency of use of these new therapies (NT) in Spain, and their impact on overall survival (OS). Methods. This is a multicenter retrospective study. Patients with DLBCL and HGBCL were identified from the RELINF platform, where GELTAMO investigators register all patients with lymphoma diagnosed in each center from January 2014. Finally, 15 centers (2 out of 15 CAR-T cell providers) accepted to participate in the study, who were asked to complete additional information related with relapse and NT. Patients were divided in 2 cohorts according to date of documentation of relapse / refractory disease: 2014-2017 (first period) and 2018-2021 (second period). The use of NT in both periods and their impact on OS were analyzed. Results We included 2162 patients in the analysis, 1891 DLBCL (51% germinal centre B-cell phenotype, 41% activated B-cell phenotype, 8% indeterminate), and 271 HGBCL (45% of them double/triple hit). Median age was 69 y/o, 72% were ≥ 70 y/o at diagnosis, and 49.8% were female. Four hundred and ninety four patients had relapsed or refractory disease, 183 in the 1st period, and 311 in the 2nd period. Median number of lines for r/r patients was 3 (2-7). NT were used in 353 patients, 30 in the 1st period and 111 in the 2nd period. New MA were used in 88 (28%) patients, being polatuzumab vedotin the most frequently used antibody (47, 54%) (tafasitamab 7, loncastuximab 5, brentuximab 11, others 10), and BA in 28 (9%), all of them within clinical trials. Twenty patients received NT in 1st relapse, 14 MA and 7 BA. From 211 patients who received > 2 lines of treatment, 128 were in the 2nd period, and 60 (47%) of them received CAR-T cell therapy, whereas 39 (30%) of these patients did not receive any NT. Five-year progression free survival (PFS) for the entire cohort was 71% (95% CI 69-74), and median overall survival (OS) was 90 months (95% CI: 76-NR). Median OS for r/r patients was 22 months (95% CI: 19 - 26), and for the group of patients who received > 2 lines was 23 months (95% CI; 19-27). With a median follow up of 25 months (50 months for the 1st period and 20 months for the 2nd period) OS was superior for patients relapsed in the 2nd period (26 months [95% CI: 22-38]), compared with those relapsed in the 1st period (18 months [IC: 16-22], p= 0.016) (Figure 1, A). Median OS since first relapse for patients pre-treated with > 2 lines who received CAR-T cells was 31 months vs 11 months for patients who did not receive CART (p= 0.002) (Figure 1, B). Conclusion: The OS of patients with r/r DLBCL and HGBCL has improved in recent years, probably in relation to the incorporation of NT, although the use of these NT in Spain has been lower than expected for this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Crebbp Kat Domain Missense Mutations Alter Its Dynamic Loading Onto Chromatin and Create a Transcription Factor Sink

The CREBBP gene encodes a lysine acetyltransferase (KAT) and is one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). However, the spectrum of mutations is significantly different between these two diseases, with significantly higher fractions of nonsense/frameshift mutations in DLBCL and comparatively higher fractions of lysine acetyltransferase (KAT) domain point mutations (KAT-PM) in FL. Among KAT domain mutations, multiple hotspots can be observed with R1446 (14%), Y1482 (3%) and Y1503 (7%) being the most frequently mutated residues. We have previously shown that CREBBP R1446C mutations lead to a more profound loss of histone acetylation and gene expression compared to biallelic knock-out (KO) of the CREBBP highlighting a potential dominant-negative function of R1446C via an unknown mechanism. We have also performed biochemical and structural analysis of KAT domain mutations and observed that R1446C mutations result in a partial loss of function (pLOF), whereas Y1482N and Y1503C mutations are catalytically dead. Here, we report the molecular characterization of CRISPR-engineered isogenic cell lines (RL, HT, 293T) bearing biallelic KO, R1446C, Y1482N or Y1503C mutations, or WT CREBBP, using CUT&RUN analysis of CREBBP and EP300 loading, histone marks (H3K27Ac, H3K4me1, H3K4me2, H3K27me3), and ATAC and RNA sequencing for chromatin accessibility and gene expression, respectively. Western blot analysis showed significant reduction of H3K27Ac and H3K18Ac in all point-mutants with no alteration of CREEBP or its paralog EP300 expression. Principal component analysis (PCA) of H3K27Ac and gene expression data revealed that KO cells were most similar to WT, and all KAT-PM cells clustered separately to WT and KO cells. Notably, R1446C (partial LOF) cells reproducibly clustered separately from Y1482N and Y1503C (catalytically dead), and differential analysis identified both shared and R1446C- or Y1482N/Y1503-specific losses of H3K27Ac in all cell lines, suggesting that the degree of loss in catalytic activity rendered by different KAT domain point mutations may be associated with distinct molecular phenotypes. In order to investigate the mechanism by which KAT-PMs drive more severe molecular changes compared to biallelic KO, we investigate the redundant role of EP300 using CUT&RUN analysis of EP300 loading. CREBBP-bound active enhancers (H3K4me1+ H3K27Ac+) showed a significant increase in EP300 loading in cells with CREBBP KO compared to WT. However, we observed a slight decrease in EP300 loading within KAT-PM cells, indicating that the presence of mutant CREBBP protein prevents the redundant activity of EP300. We speculated that this may be due to mutant CREBBP residing on chromatin in an inactive state and preventing loading of EP300. We therefore analyzed CREBBP loading over CREBBP-bound active enhancers in WT cells that had significant reductions of H3K27Ac in KAT-PMs. Surprisingly, we observed a significant reduction in CREBBP loading over these regions, with a greater loss of CREBBP loading in catalytically dead mutants (Y1482N/Y1503C) compared to the partial LOF mutant (R1446C). We therefore characterized patterns of CREBBP loading during normal germinal center B-cell development by analyzing purified tonsillar naïve B-cells, centroblasts and centrocytes. We observed that CREBBP is dynamically loaded onto chromatin during the centroblast-to-centrocyte transition, indicating that loss of chromatin-bound CREBBP in KAT-PMs may be due to a failure of mutant CREBBP to load onto these sites. We next performed transcription factor (TF) motif enrichment analysis and identified a significant enrichment of key TFs such as BACH2 and IRF4 in regions with failed CREBBP loaded and EP300 compensation, indicating that CREBBP may bind these transcription factors and act as a sink that prevents redundant EP300 activity. We validated this using tetracycline-inducible over-expression of IRF4 in KAT-PM cells, which increased EP300 loading and H3K27Ac over IRF4 binding sites. Collectively, our results indicate that CREBBP KAT-PMs prevent the redundant activity of EP300 in a manner proportionally to their relative loss of catalytic activity, by preventing the dynamic loading of CREBBP onto chromatin and acting as a transcription factor sink.

Functional Profiling Identifies Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Diffuse Large B-Cell Lymphoma

Strong expression of MYC and MYC-regulated genes defines the molecular high-grade / double-hit signature subgroup of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) which shows poor clinical outcomes with current therapy. About half of such cases show large-scale chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, but there is little understanding of the specific regulatory elements that activate MYC in absence of a rearrangement, or in the setting of non-immunoglobulin MYC rearrangement partners (RPs). We used high-throughput CRISPR-interference functional profiling (CRISPRi) to pinpoint cis-regulatory element dependencies in MYC-intact and MYC-rearranged lymphoma cell lines at the level of individual nucleosome-free regions (NFRs). Custom sgRNA libraries targeted 233 individual NFRs (median 42 sgRNA / NFR after stringent quality filtering) across the MYC topological domain (TAD) and MYC RP loci in six GCB-DLBCL cell lines and two comparator mantle cell lymphoma (MCL) cell lines. Promoter-targeting sgRNAs against 587 sequence-specific transcription factor (TF) genes and 65 essential gene controls allowed for interrogation of trans-factor dependencies in the same assay. CRISPRi profiling in one GCB-DLBCL and one MCL cell line with intra-chromosomal rearrangements of chromosome 8 showed strong, unique dependencies on enhancers within the cell-line-specific RPs, supporting an "enhancer-hijacking" function of these rearrangements. Three profiled cell lines showed structurally distinct variants of a recurrent t(3;8)(q27;q24) rearrangement that links the MYC gene to the distal super-enhancer region 150-240 kB upstream of BCL6. Although the distal BCL6 super-enhancers have been shown to sustain the essential BCL6 oncogene itself, we surprisingly found only weak dependency on the distal BCL6 enhancers for the three GCB-DLBCL cell lines lacking t(3;8) rearrangements. In contrast, all three GCB-DLBCL cell lines with t(3;8) rearrangements showed strong dependency on specific NFRs within the distal BCL6 super-enhancer. Validation assays showed that concurrent targeting of two BCL6 enhancer NFRs lead to even greater growth suppression and loss of MYC expression in t(3;8)-bearing cell lines. In both GCB-DLBCL cell lines lacking MYC rearrangements, we identified a strong, novel dependency on a specific enhancer ("GCB-ME1") in the 3' portion of the MYC TAD. This enhancer was acetylated in MYC-intact GCB-DLBCL patient samples and normal centroblasts, but not in MCL or MYC-rearranged DLBCL samples. We confirmed that sustained MYC expression in MYC-intact cell lines requires activity of GCB-ME1. GCB-ME1 showed little or no essentiality in the four MYC-rearranged DLBCL cell lines or in the MYC-intact MCL cell line SP-49, which is dependent on previously identified Notch/RBPJ-regulated 5' MYC enhancers (Ryan et al., 2017). ChIP-Seq showed that both GCB-ME1 and all of the most strongly essential BCL6 enhancers in t(3;8) cell lines were strongly bound by a triad of TFs, MEF2B, POU2F2, and POU2AF1, that were previously implicated in BCL6 activation. These genes were required to sustain MYC expression in both MYC-intact and t(3;8) GCB-DLBCL, and were highly essential in those cell lines, but POU2F2 and MEF2B were not essential in some cell lines with alternate MYC rearrangements. Our data show that DLBCL is highly sensitive to selective disruption of discrete MYC-activating enhancers. Transcriptional regulatory factor dependencies in DLBCL may be shaped by the requirement to sustain MYC activation via the native GCB-ME1 enhancer or alternately via enhancers in specific RP loci, a finding with implications for therapeutic targeting of transcriptional regulatory mechanisms in DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First-in-Class Histone Acetyltransferase (HAT) Activator, YF2, Modulates Immune Evasion in DLBCL, Enhancing the Effects of Immune Checkpoint Blockade

We reported the discovery of YF2, a EP300 (p300) and CREBBP (CBP) HAT activator which demonstrates selective cytotoxicity in HAT-mutated DLBCL cell lines and induces histone acetylation in vitro and in vivo. Recently, we have focused on the role of HAT induction in regulating immune response in DLBCLs. CBP has been shown to function as an enhancer that regulates GC B-cell fate by controlling antigen presentation and terminal differentiation. HAT mutations are linked to reduced MHC-II HLA transcripts due to decreased acetylation at the promoter regions which leads to decreased GC B:T cell entanglement and derangements in the GC reaction. Numerous studies have reported the effect of epigenetic drugs on antigen presentation. This response is caused by disruption of the BLC6/SMRT/HDAC3 repressor complex which co-occupies enhancers in the MHC Class II loci. Previously, we demonstrated YF2 induced p300 mediated acetylation of BCL6; abrogating its activity. More recently, we demonstrated through RNA-seq that prolonged treatment of YF2 upregulated the expression of several MHC Class I-II genes resulting in the upregulation of the immune regulation signaling pathway. Further, treatment with YF2 up-regulated MHC-I and -II expression in a dose dependent manner. In addition to regulating immune response, BCL6 is a master regulator of GC formation. We hypothesize that induction of HATs will modulate immune response in splenic and circulating B and T-cells. Immunized BALB/c mice were treated with saline or 50mg/kg YF2 for 10 days. Spleen and blood samples were analyzed by flow cytometry. We observed a significant reduction in splenic weight, B-cell frequency, and GCs formation in YF2 treated mice. YF2 treatment increased the frequency of mature and transitional B-cells in the spleen. Further, YF2 treatment reduced light zone and increased dark zone cells in the spleen. YF2 treatment also increased the frequency of plasma cells and plasmablast. MHC-I and -II expression was significantly upregulated in YF2 treated mice. YF2 exposure resulted in an increase of PD-L1hi B-cells. Lastly, circulating B-cells showed significant increase in MHC-II expression following YF2 treatment. In splenic T-cells, we observed a reduction in CD4+ helper T-cells and an increase in CD8+ cytotoxic T-cells in YF2 treated mice. YF2 caused a reduction in naïve CD4+ cells and an increase in the frequency of central and effector memory cells Lastly, we examined the frequency of inhibitory molecules PD1, TIGIT, CD226, Lag3, and VISTA. PD-1, TIGIT, and CD226 were upregulated with YF2 treatment in CD4+ cells and CD226 was upregulated in CD8+ cells. Together, these results demonstrate YF2 is able to modulate immune response in B and T-cells. DLBCLs are known to have limited sensitivity to checkpoint blockade with PD-1 and PD-L1 inhibitors due to low PD-L1 and PD-1 expression which is in part driven by HAT mutations and overexpression of BCL6; which regulates PD-L1. We hypothesize that combination treatment of YF2 and PD-1 inhibitor in the immunocompetent syngeneic A20 mouse tumor xenograft model will result in decreased tumorigenicity and improved survival in mice. Xenografted and immunized BALBc mice were treated with saline, YF2, PD-1 inhibitor, or a combination of YF2 and PD-1 inhibitor for 28 days. We observed, a significant reduction in tumor volume in both the PD-1 and combination treated groups. Kaplan-Meier analysis showed an increase in median survival for all treatment with the combination treatment resulting in the greatest increase in median survival vs. control (Fig A). Spatial analysis was performed using the 10x Visium platform and sc-RNA-seq to examine the tumor microenvironment in control and YF2 treated mouse tumor samples (Fig B). We observed a more aggressive phenotype in the untreated tumors as well as an increase in the number of distinct clusters indicating a more heterogenous tumor population. Lastly, PBMCs from patient samples were isolated and treated with YF2. Compared to healthy donor patients, YF2 induced cytotoxicity across various lymphoma subtype. Normal PBMCs were not affected by treatment. Based on the success of YF2 in the preclinical setting, the use of this first-in-class drug for treatment of lymphoma was approved to proceed in clinical studies in patients with relapsed/refractory lymphoma. Patient samples will be collected and analyzed for immune response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Advances in Understanding of Metabolism of B-Cell Lymphoma: Implications for Therapy.

There have been significant recent advances in the understanding of the role of metabolism in normal and malignant B-cell biology. Previous research has focused on the role of MYC and mammalian target of rapamycin (mTOR) and how these interact with B-cell receptor signaling and hypoxia to regulate glycolysis, glutaminolysis, oxidative phosphorylation (OXPHOS) and related metabolic pathways in germinal centers. Many of the commonest forms of lymphoma arise from germinal center B-cells, reflecting the physiological attenuation of normal DNA damage checkpoints to facilitate somatic hypermutation of the immunoglobulin genes. As a result, these lymphomas can inherit the metabolic state of their cell-of-origin. There is increasing interest in the potential of targeting metabolic pathways for anti-cancer therapy. Some metabolic inhibitors such as methotrexate have been used to treat lymphoma for decades, with several new agents being recently licensed such as inhibitors of phosphoinositide-3-kinase. Several other inhibitors are in development including those blocking mTOR, glutaminase, OXPHOS and monocarboxylate transporters. In addition, recent work has highlighted the importance of the interaction between diet and cancer, with particular focus on dietary modifications that restrict carbohydrates and specific amino acids. This article will review the current state of this field and discuss future developments.

MTOR inhibition amplifies the anti-lymphoma effect of PI3Kβ/δ blockage in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kβ/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell–like (ABC) and germinal center B-cell–like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kβ/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kβ/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kβ/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kβ/δ signaling and thus identifies a previously unappreciated role of the PI3Kβ isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kβ/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

Single-cell profiling reveals a memory B cell-like subtype of follicular lymphoma with increased transformation risk

Follicular lymphoma (FL) is an indolent cancer of mature B-cells but with ongoing risk of transformation to more aggressive histology over time. Recurrent mutations associated with transformation have been identified; however, prognostic features that can be discerned at diagnosis could be clinically useful. We present here comprehensive profiling of both tumor and immune compartments in 155 diagnostic FL biopsies at single-cell resolution by mass cytometry. This revealed a diversity of phenotypes but included two recurrent patterns, one which closely resembles germinal center B-cells (GCB) and another which appears more related to memory B-cells (MB). GCB-type tumors are enriched for EZH2, TNFRSF14, and MEF2B mutations, while MB-type tumors contain increased follicular helper T-cells. MB-type and intratumoral phenotypic diversity are independently associated with increased risk of transformation, supporting biological relevance of these features. Notably, a reduced 26-marker panel retains sufficient information to allow phenotypic profiling of future cohorts by conventional flow cytometry.

Distinct B-Cell Specific Transcriptional Contexts of the BCL2 Oncogene Impact Pre-Malignant Development in Mouse Models.

Upregulated expression of the anti-apoptotic BCL2 oncogene is a common feature of various types of B-cell malignancies, from lymphoma to leukemia or myeloma. It is currently unclear how the various patterns of deregulation observed in pathology eventually impact the phenotype of malignant B cells and their microenvironment. Follicular lymphoma (FL) is the most common non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells, and its major hallmark is the t(14:18) translocation occurring in B cell progenitors and placing the BCL2 gene under the control of the immunoglobulin heavy chain locus regulatory region (IgH 3'RR), thus exposing it to constitutive expression and hypermutation. Translocation of BCL2 onto Ig light chain genes, BCL2 gene amplification, and other mechanisms yielding BCL2 over-expression are, in contrast, rare in FL and rather promote other types of B-cell lymphoma, leukemia, or multiple myeloma. In order to assess the impact of distinct BCL2 deregulation patterns on B-cell fate, two mouse models were designed that associated BCL2 and its full P1-P2 promoter region to either the IgH 3'RR, within a "3'RR-BCL2" transgene mimicking the situation seen in FL, or an Ig light chain locus context, through knock-in insertion at the Igκ locus ("Igκ-BCL2" model). While linkage to the IgH 3' RR mostly yielded expression in GC B-cells, the Igκ-driven up-regulation culminated in plasmablasts and plasma cells, boosting the plasma cell in-flow and the accumulation of long-lived plasma cells. These data demonstrate that the timing and level of BCL2 deregulation are crucial for the behavior of B cells inside GC, an observation that could strongly impact the lymphomagenesis process triggered by secondary genetic hits.

A bone-based 3D scaffold as an in-vitro model of microenvironment-DLBCL lymphoma cell interaction.

About 30% of patients with diffuse large B-cell lymphoma (DLBCL) relapse or exhibit refractory disease (r/r DLBCL) after first-line immunochemotherapy. Bone marrow (BM) involvement confers a dismal prognosis at diagnosis, likely due to the interaction between neoplastic cells and a complex tumor microenvironment (TME). Therefore, we developed a 3D in-vitro model from human decellularized femoral bone fragments aiming to study the role of mesenchymal stromal cells (MSC) and the extracellular matrix (ECM) in the adaptation, growth, and drug resistance of DLBCL lymphoma cells. The 3D spatial configuration of the model was studied by histological analysis and confocal and multiphoton microscopy which allowed the 3D digital reproduction of the structure. We proved that MSC adapt and expand in the 3D scaffold generating niches in which also other cell types may grow. DLBCL cell lines adhered and grew in the 3D scaffold, both in the presence and absence of MSC, suggesting an active ECM-lymphocyte interaction. We found that the germinal center B-cell (GCB)-derived OCI-LY18 cells were more resistant to doxorubicin-induced apoptosis when growing in the decellularized 3D bone scaffold compared to 2D cultures (49.9% +/- 7.7% Annexin V+ cells in 2D condition compared to 30.7% + 9.2% Annexin V+ 3D adherent cells in the ECM model), thus suggesting a protective role of ECM. The coexistence of MSC in the 3D scaffold did not significantly affect doxorubicin-induced apoptosis of adherent OCI-LY18 cells (27.6% +/- 7.3% Annexin V+ 3D adherent cells in the ECM/MSC model after doxorubicin treatment). On the contrary, ECM did not protect the activated B-cell (ABC)-derived NU-DUL-1 lymphoma cell line from doxorubicin-induced apoptosis but protection was observed when MSC were growing in the bone scaffold (40.6% +/- 5.7% vs. 62.1% +/- 5.3% Annexin V+ 3D adherent cells vs. 2D condition). These data suggest that the interaction of lymphoma cells with the microenvironment may differ according to the DLBCL subtype and that 2D systems may fail to uncover this behavior. The 3D model we proposed may be improved with other cell types or translated to the study of other pathologies with the final goal to provide a tool for patient-specific treatment development.

A t(11;14)(q13;q32)/CCND1::IGH carrying progenitor germinal B-cell with subsequent cytogenetic aberrations contributes to the development of classic Hodgkin lymphoma

Classic Hodgkin lymphoma (cHL) is characterized by the presence of Hodgkin Reed-Sternberg (HRS) cells. Although HRS cells express PAX5, cHL frequently lacks other B-cell markers. There is now evidence that HRS cells are monoclonal and are derived from germinal center B-cells. In terms of genetic aberrations, cHL frequently exhibit activated NF-kB signaling pathway. In this study, we present a case of cHL harboring a t(11;14) (q13;q32)/CCND1::IGH, identified by chromosome and fluorescence in situ hybridization analysis and with CCND1 expression in HRS cells. We also analyzed recurrent cytogenetic aberrations in t(11;14) positive mantle cell lymphoma (MCL) and those found in cHL from the literature to assess genetic overlap, clonal evolution, and to identify potential signaling pathways in cHL with CCND1::IGH. This analysis suggests the development of t(11;14)+ cHL and MCL from a transformed precursor cell with t(11;14) through genetic evolution and consequent deregulated pathways, including the NF-κB and NOTCH1 signaling.

Clinical characteristics and prognosis of patients with co-existing follicular lymphoma and diffuse large B-cell lymphoma components in rituximab era.

This single-centre study aimed to determine the clinicopathological characteristics and prognosis for patients with co-existing FL and DLBCL components (FL/DLBCL). We retrospectively analysed the clinical characteristics and prognosis of patients diagnosed with FL/DLBCL (n = 56) and with pure FL (n = 260) or de novo DLBCL (n = 812) (controls) between January 2013 and December 2021. The median age of patients with FL/DLBCL was 52years. The amount of the DLBCL component ranged from 5 to 95%. Among the 56 FL/DLBCL cases analysed, 67.9% were of germinal centre B-cell (GCB) origin, 26.8% non-GCB origin, and 5.3% were unclassified. The clinical features of patients with FL/DLBCL were intermediate, falling between those of FL and DLBCL. Propensity-score matching was performed for patients with similar baseline characteristics who were receiving the rituximab plus cyclophosphamide, doxorubicin or epirubicin, vindesine, and prednisone (R-CHOP) regimen. Patients with FL/DLBCL showed inferior outcomes compared to those with FL, with a lower complete remission (CR) rate, progression-free survival (PFS), and overall survival (OS). Bone marrow involvement and B symptoms were identified as independent adverse prognostic factors for PFS among patients with FL/DLBCL. Patients with FL/DLBCL presented a lower CR rate and PFS but similar OS to those with DLBCL when receiving the R-CHOP regimen. Patients with FL/DLBCL showed inferior treatment response and survival than those with pure FL and had a lower CR rate and PFS, but similar OS to those with DLBCL in the rituximab era.

The multilobated morphology is still a better prognosis factor of diffuse large B-cell lymphoma in the R-CHOP era.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of B-cell lymphoma. Although the multilobated subtype of DLBCL has been observed since the 1970s, little is known about the clinical significance of this unique variant in the era of rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone/prednisolone (R-CHOP) therapy. In this study, the retrospective clinicopathological analysis of 312 patients diagnosed with DLBCL showed that the multilobated DLBCL group comprised 11% of the cases and was predominantly male (p = 0.027), achieved complete remission in the first therapy (p = 0.023), and exhibited germinal center B-cell phenotypes in the Hans algorithm (p = 0.025). The multilobated DLBCL groups had a better prognosis in overall survival (OS) and progression-free survival (PFS) than the non-multilobated DLBCL group (OS, p = 0.006; PFS, p = 0.010). In the multivariate Cox regression analyses for OS, independent prognosis factors were high soluble IL-2 receptor (p = 0.025), high risk of International Prognostic Index, and multilobated morphology (p = 0.031). The most characteristic copy number gains found in more than 50% of the cases were located at 1q, 3p, 10q, 12q, and 14q. Overall, the multilobated morphology in DLBCL exhibits a good outcome in the R-CHOP era.

Deciphering imprints of impaired memory B-cell maturation in germinal centers of three patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID), characterized by recurrent infections, low serum class-switched immunoglobulin isotypes, and poor antigen-specific antibody responses, comprises a heterogeneous patient population in terms of clinical presentation and underlying etiology. The diagnosis is regularly associated with a severe decrease of germinal center (GC)-derived B-cell populations in peripheral blood. However, data from B-cell differentiation within GC is limited. We present a multiplex approach combining histology, flow cytometry, and B-cell receptor repertoire analysis of sorted GC B-cell populations allowing the modeling of distinct disturbances in GCs of three CVID patients. Our results reflect pathophysiological heterogeneity underlying the reduced circulating pool of post-GC memory B cells and plasmablasts in the three patients. In patient 1, quantitative and qualitative B-cell development in GCs is relatively normal. In patient 2, irregularly shaped GCs are associated with reduced somatic hypermutation (SHM), antigen selection, and class-switching, while in patient 3, high SHM, impaired antigen selection, and class-switching with large single clones imply increased re-cycling of cells within the irregularly shaped GCs. In the lymph nodes of patients 2 and 3, only limited numbers of memory B cells and plasma cells are formed. While reduced numbers of circulating post GC B cells are a general phenomenon in CVID, the integrated approach exemplified distinct defects during GC maturation ranging from near normal morphology and function to severe disturbances with different facets of impaired maturation of memory B cells and/or plasma cells. Integrated dissection of disturbed GC B-cell maturation by histology, flow cytometry, and BCR repertoire analysis contributes to unraveling defects in the essential steps during memory formation.

ABCL-523 Immunohistochemical (IHC) Expression of Epigenetic Regulators (EZH-2 and H3K27) as well as Cell Cycle Regulators (P16, P53 and RB) in Patients Diagnosed With Primary Diffuse Large B-Cell Lymphoma (DLBCL) and Association With Therapy Response Rates and Survival

Despite advancements in chemotherapeutics, survival rate in DLBCL remains 50% in developing countries. Transcriptomic profiling has shown that EZH-2 has a role in germinal center B-Cell (GCB) type of DLBCL, NOS. To evaluate IHC expression of epigenetic regulators and cell cycle regulators in DLBCL, NOS and correlate with cell of origin (GCB type and non-GCB type), therapy response and patient survival. A retrospective study of patients diagnosed with primary DLBCL, NOS in 2014-2015 who were followed-up until April 2022. DLBCL was classified by Han's Algorithm and IHC for EZH-2, H3K27, P16, P53, RB was done & scored semi-quantitatively. Various clinical parameters at baseline as well as treatment modalities given were also noted. Study conducted at a tertiary referral cancer care center on patients visiting the out-patient department. Patients were selected continuously from the out-patient records of the Department of Medical Oncology during 2014-2015. 37 patients were included, out of which follow-up data was available for 21 patients. Association between IHC expression and various clinical parameters as well as correlation with cell of origin, therapy response and patient survival. Out of 37 patients, 62.16% were male, with male to female ratio of 1.64. Age (48.6±14.1) ranged from 22-78 years. 15/37(40.5%) patients were diagnosed as DLBCL-GCB type. 18/37(48.6%) patients presented with at least one B-symptom. 20/37(54%) patients presented at higher stage of disease (3 or 4), while 17/37(46%) had extra-nodal involvement. Derived neutrophil-lymphocyte ratio (dNLR) was ≥ 2.2 in 13/37(35%) patients. Association of H3K27 with RB(p=0.024) and EZH-2 with P53(p=0.001) was found to be statistically significant. No other significant association was found between the various markers as well as with the cell of origin. However, association of H3K27 with response rate (RR)(p=0.012) and EZH-2 with RR(p=0.028) was found to be statistically significant. EZH-2 pathway has been described in GCB type of DLBCL. Though IHC expression contributed to a statistically significant difference in RR, the expression was not limited to GCB subtype as defined by Han's Algorithm. Therefore, further larger studies are required for validation of IHC based expression. AIIMS, Delhi.

NF-κB Mutations in Germinal Center B-Cell Lymphomas: Relation to NF-κB Function in Normal B Cells.

Most B cell lymphomas arise from the oncogenic transformation of B cells that have undergone the germinal center (GC) reaction of the T cell-dependent immune response, where high-affinity memory B cells and plasma cells are generated. The high proliferation of GC B cells coupled with occasional errors in the DNA-modifying processes of somatic hypermutation and class switch recombination put the cell at a risk to obtain transforming genetic aberrations, which may activate proto-oncogenes or inactivate tumour suppressor genes. Several subtypes of GC lymphomas harbor genetic mutations leading to constitutive, aberrant activation of the nuclear factor-κB (NF-κB) signaling pathway. In normal B cells, NF-κB has crucial biological roles in development and physiology. GC lymphomas highjack these activities to promote tumour-cell growth and survival. It has become increasingly clear that the separate canonical and non-canonical routes of the NF-κB pathway and the five downstream NF-κB transcription factors have distinct functions in the successive stages of GC B-cell development. These findings may have direct implications for understanding how aberrant NF-κB activation promotes the genesis of various GC lymphomas corresponding to the developmentally distinct GC B-cell subsets. The knowledge arising from these studies may be explored for the development of precision medicine approaches aimed at more effective treatments of the corresponding tumours with specific NF-κB inhibitors, thus reducing systemic toxicity. We here provide an overview on the patterns of genetic NF-κB mutations encountered in the various GC lymphomas and discuss the consequences of aberrant NF-κB activation in those malignancies as related to the biology of NF-κB in their putative normal cellular counterparts.

INCIDENTAL PRIMARY PULMONARY DIFFUSE LARGE B-CELL LYMPHOMA

INCIDENTAL PRIMARY PULMONARY DIFFUSE LARGE B-CELL LYMPHOMA

Overexpression of interleukin-20 correlates with favourable prognosis in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma worldwide, accounting for about 40% of cases. The role of cytokines in the pathogenesis of lymphomas has been rarely addressed, although cytokines have a close immunological relationship with lymphocytes. We observed overexpression of interleukin (IL)-20 in reactive germinal centres (GCs) leading us to hypothesise that IL-20 may play a role in lymphomagenesis. In this study, we surveyed for IL-20 expression in various types of lymphoma and found that IL-20 was expressed most frequently in follicular lymphoma (94%), but also in Burkitt lymphoma (81%), mantle cell lymphoma (57%), nodal marginal zone lymphoma (56%), Hodgkin lymphomas (50%), small lymphocytic lymphoma (50%) and diffuse large B-cell lymphoma (DLBCL, 48%). IL-20 was not expressed in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), lymphoplasmacytic lymphoma, and plasmacytoma. T-cell lymphomas were largely negative for IL-20 expression, except for anaplastic large cell lymphoma (ALCL, 61%), which frequently expressed IL-20, especially in cutaneous ALCL, and showed an inverse association with ALK expression (p=0.024). We further tested IL-20 expression in another large cohort of DLBCL and found IL-20 expression more frequently in germinal centre B-cell (GCB) than in non-GCB subtype [16/26 (62%) versus 24/64 (38%), p=0.038]. In this cohort, IL-20 was associated with a lower rate of extranodal involvement (p=0.009), bone marrow involvement (p=0.040), and better overall survival (p=0.020). Mechanistically, IL-20 overexpression promoted G1 cell cycle arrest and subsequent apoptosis of DLBCL cells and vice versa invitro. We conclude that IL-20 may be involved in lymphomagenesis and may be useful as a prognostic marker in patients with DLBCL. In addition, IL-20 plays an inhibitory role in DLBCL growth, probably through cell cycle regulation.

Abstract A15: Clinico-pathologic and molecular characteristics of primary extranodal germinal-center B-cell like diffuse large B-cell lymphoma (EN GCB-DLBCL)

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease and in 30-40% cases can present or relapse at extranodal sites. Primary extranodal DLBCL (EN-DLBCL) is defined as disease presenting as clinically dominant extranodal masses with no or only minor nodal disease. EN-DLBCL is predominantly of the activated B-cell (ABC) and less commonly of germinal center B-cell (GCB) like DLBCL subtype. We aimed to identify genetic characteristics of EN GCB-DLBCL to gain insights into extranodal tropism and aggressive clinical behavior. Methods: Patient data was collected via retrospective chart review. Inclusion criteria consisted of pathologically confirmed diagnosis of GCB-DLBCL by the Hans algorithm, extra-nodal involvement at diagnosis or relapse, and availability of next-generation sequencing (NGS). Overall (OS) and progression-free survival (PFS) were analyzed using the Kaplan-Meier method. Results: Fifteen patients were identified. The median age at diagnosis was 64 years (range 33-89) and majority were men (62.5%). Two patients had purely nodal disease at presentation that relapsed at EN sites, while the remainder (13/15) had predominantly EN disease. EN sites included central nervous system [CNS] (n = 5), bone marrow (n = 4), epidural (n=4), bone (2), paranasal sinus (1), testes (1), stomach (n = 2), lung/pleura (n=2), and liver (n=3). Nine patients had disease in multiple EN sites. All patients had advanced stage disease and median IPI score was 3 (range 3-4). On FISH/cytogenetic analysis, two patients had MYC and four patients had BCL2 rearrangements. Copy gains in BCL6, BCL2 and MYC were seen in 2, 1 and 4 patients respectively. No patients had evidence of MYC and BCL2 and/or BCL6 rearrangements (double hit). TP53 deletion was seen in 1 patient. On NGS, prominent gene mutations identified were KMTD2 (8/15, 53%), TP53 (5/15, 33%), TNFRSF14 (4/15, 27%), and evidence of aneuploidy with abnormalities in chromosome 12 (5/15, 33%), chromosome 6 (4/15, 27%), and chromosome 8 (4/15, 27%), including both losses and gains of function. Notably, MYD88 (3/15, 20%), CARD11 (2/15, 13%), and IRF4 (2/15, 15%) mutations, representative of BCR/TLR4 pathway activation and NF-kB signaling were seen at initial presentation. All patients were treated with chemo-immunotherapies incorporating CNS prophylaxis and 2 patients received salvage CAR T-cell therapy. Median OS was 1.3 years and median PFS was 11.3 months. Nine patients (63%) progressed, with CNS relapses in 5 and of these, 7 patients died. Conclusions: This study shows that EN GCB-DLBCL is molecularly similar to EN ABC-DLBCL; specifically, activation of NF-kB pathway signaling through gene mutations involving the MCD (MyD88/CD79B) cluster is prominent, and this potentially enables extranodal tropism. The complex karyotype and TP53 mutations may contribute to chemoresistance and aggressive disease biology. Our results provide valuable molecular information and can aid in personalizing treatment strategies for this uncommon, aggressive DLBCL subset. Citation Format: Akshat Patel, Brian Lue, Kiran A Kumar, Farrukh T Awan, Jeffrey Gagan, Weina Chen, Praveen Ramakrishnan Geethakumari. Clinico-pathologic and molecular characteristics of primary extranodal germinal-center B-cell like diffuse large B-cell lymphoma (EN GCB-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 A15.