Germinal Center B-cell Research Articles

The FBXO45-GEF-H1 axis controls germinal center formation and B-cell lymphomagenesis.

The role of ubiquitin-mediated degradation mechanisms in the pathogenesis of diffuse large B cell (DLBCL) and follicular lymphoma (FL) is not completely understood. We show that conditional deletion of the E3 ubiquitin ligase Fbxo45 in germinal center B-cells results in B-cell lymphomagenesis in homozygous (100%) and heterozygous (48%) mice. Mechanistically, FBXO45 targets the RHO guanine exchange factor ARHGEF2/GEF-H1 for ubiquitin-mediated degradation. Double genetic ablation of Fbxo45 and Arhgef2 ameliorated lymphoma formation. Transgenic knock-in mice harboring a GEF-H1 mutant unable to bind FBXO45 develop B-cell lymphomas with ~50% penetrance. Genome sequencing in human lymphomas identified mutually-exclusive FBXO45 copy number losses and ARHGEF2 gains, with combined frequencies ranging from 26.32% in FL to 45.12% in DLBCL. Notably, FBXO45 silencing enhances sensitivity to MEK1/2 inhibition. These results identify FBXO45 and ARHGEF2 as a novel tumor-suppressor and oncogene pair involved in the pathogenesis of B-cell lymphomas with significant implications for targeted therapies.

Diffuse Large B-Cell Lymphoma/High Grade B-Cell Lymphoma with MYC and BCL6 Rearrangements: a study of 60 Cases.

Classification of cases of diffuse large B-cell lymphoma (DLBCL)/high-grade B-cell lymphoma (HGBL) with MYC and BCL6 rearrangements, also known as BCL6 double hit lymphoma (DHL), is controversial. We assessed 60 cases of BCL6-DHL and compared this cohort to 224 cases of DHL with MYC and BCL2 rearrangements (BCL2-DHL) and 217 cases of DLBCL not otherwise specified. Compared with the DLBCL cohort, BCL6-DHL patients had more aggressive clinical features such as frequent extranodal involvement, high-stage disease, high IPI score and elevated serum lactate dehydrogenase level (p <0.01 for all). Compared with the BCL2-DHL cohort, BCL6-DHL patients had similarly aggressive clinical features but a lower frequency of germinal center B-cell (GCB) immunophenotype and MYC and BCL2 double expression. Patients with BCL6-DHL showed an overall survival (OS) intermediate between patients with DLBCL and BCL2-DHL. Following induction with R-CHOP chemotherapy, BCL6-DHL patients demonstrated a poor OS similar to BCL2-DHL patients and worse than that of DLBCL patients (p = 0.024). However, among patients who received R-EPOCH, there was no significant difference in OS among the three groups (p = 0.146). Gene expression profiling showed that 60% of BCL6-DHL cases had a double hit (DH)-like signature compared with 10% of DLBCL-GCB and 93% of BCL2-DHL. The DH-like signature in BCL6-DHL cases was associated with a GCB immunophenotype. Based on these data, we suggest that BCL6-DHL cases are clinically more aggressive than DLBCL and patients may benefit from a more aggressive therapy than R-CHOP. The data also suggest that BCL6-DHL as currently defined, is heterogeneous and that neoplasms with a GCB immunophenotype are more likely to have DH-like signature and behave more aggressively. Lastly, we suggest that BCL6-DHL cases deserve to be recognized separately in a lymphoma classification to facilitate further understanding of these neoplasms and for optimal patient management.

Targeting DOT1L and EZH2 synergizes in breaking the germinal center identity of Diffuse Large B-cell Lymphoma.

Differentiation of antigen-activated B cells into pro-proliferative germinal center (GC) B cells depends on the activity of the transcription factors MYC and BCL6, and the epigenetic writers DOT1L and EZH2. GCB-like Diffuse Large B Cell Lymphomas (GCB-DLBCLs) arise from GCB cells and closely resemble their cell of origin. Given the dependency of GCB cells on DOT1L and EZH2, we investigated the role of these epigenetic regulators in GCB-DLBCLs and observed that GCB-DLBCLs synergistically depend on the combined activity of DOT1L and EZH2. Mechanistically, inhibiting both enzymes led to enhanced derepression of PRC2 target genes compared to EZH2 single treatment, along with the upregulation of BCL6 target genes and suppression of MYC target genes. The sum of all these alterations results in a 'cell identity crisis', wherein GCB-DLBCLs lose their pro-proliferative GC identity and partially undergo PC differentiation, a state associated with poor survival. In support of this model, combined epi-drugging of DOT1L and EZH2 prohibited the outgrowth of human GCB-DLBCL xenografts in vivo. We conclude that the malignant behavior of GCB-DLBCLs strongly depends on DOT1L and EZH2 and that combined targeting of both epigenetic writers may provide an alternative differentiation-based treatment modality for GCB-DLBCL.

Characterization of tumor microenvironment and cell interaction patterns in testicular and diffuse large B-cell lymphomas.

The tumor microenvironments (TME) of diffuse large B-cell lymphoma (DLBCL) subgroups have remained poorly characterized. Here, we dissected the composition and spatial organization of the TME in germinal center B-cell (GCB), activated B-cell (ABC), and testicular DLBCLs (T-DLBCL) using gene expression profiling and multiplex immunohistochemistry. We found that high proportions of M2-like tumor-associated macrophages (TAMs) and cytotoxic tumor-infiltrating T cells (TILs) were characteristic of ABC DLBCL TME. Furthermore, high CD8+ TIL content translated to favorable outcomes. In contrast, GCB DLBCL TME was enriched in CD4+ TILs, regulatory TILs, and a higher M1-like/M2-like TAM ratio, and high proportions of TAMs and Granzyme B+ cells associated with worse survival. TILs and TAMs interacted more frequently with M2-like TAMs and cytotoxic TILs in the ABC DLBCLs contrary to GCB subtype, where the interactions were more abundant with other TILs and CD4+ TILs. In T-DLBCL, TME resembled that of ABC DLBCL with a higher proportion of M2-like TAMs and cytotoxic cells, except that checkpoint-positive TILs were less prominent compared to DLBCL NOS. Cytotoxic TILs also interacted more with TILs and TAMs. A high amount of CD163+ TAM interactions with distinct TILs translated to unfavorable survival both in GCB DLBCL and T-DLBCL, whereas a high number of interactions between TILs and TAMs, CD4+ TILs and TAMs, and CD4+ TILs and other TILs associated with favorable outcomes only in T-DLBCL. Together, our data demonstrate biologically and clinically relevant differences in the composition of and cellular interactions in the TME between various DLBCL entities.

Epi-microRNA mediated metabolic reprogramming counteracts hypoxia to preserve affinity maturation

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centers (GCs). Among these, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by utilizing energy provided by oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)−155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Mechanistically, miR-155 regulates H3K36me2 levels in hypoxic conditions by directly repressing the histone lysine demethylase, Kdm2a, whose expression increases in response to hypoxia. The miR-155-Kdm2a interaction is crucial for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia, thereby preventing excessive production of reactive oxygen species and subsequent apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity GC-B cells, ensuring their expansion and consequently affinity maturation.

DGKα and ζ Deficiency Causes Regulatory T-Cell Dysregulation, Destabilization, and Conversion to Pathogenic T-Follicular Helper Cells to Trigger IgG1-Predominant Autoimmunity.

Regulatory T cells (Tregs) actively engage in immune suppression to prevent autoimmune diseases but also inhibit anti-tumor immunity. Although Tregs express a TCR repertoire with relatively high affinities to self, they are normally quite stable and their inflammatory programs are intrinsically suppressed. We report here that diacylglycerol (DAG) kinases (DGK) ( and ( are crucial for homeostasis, suppression of proinflammatory programs, and stability of Tregs and for enforcing their dependence on CD28 costimulatory signal. Treg-specific deficiency of both DGK( and ( derails signaling, metabolic, and transcriptional programs in Tregs to cause dysregulated phenotypic and functional properties and to unleash conversion to pathogenic exTregs, especially exTreg-T follicular helper (Tfh) 2 cells, leading to uncontrolled effector T cell differentiation, deregulated germinal center (GC) B-cell responses and IgG1/IgE predominant antibodies/autoantibodies, and multiorgan autoimmune diseases. Our data not only illustrate the crucial roles of DGKs in Tregs to maintain self-tolerance but also unveil a Treg-to-self-reactive-pathogenic-exTreg-Tfh-cell program that is suppressed by DGKs and that could exert broad pathogenic roles in autoimmune diseases if unchecked.

Modulation of antigen delivery and lymph node activation in nonhuman primates by saponin adjuvant saponin/monophosphoryl lipid A nanoparticle.

Saponin-based vaccine adjuvants are potent in preclinical animal models and humans, but their mechanisms of action remain poorly understood. Here, using a stabilized HIV envelope trimer immunogen, we carried out studies in nonhuman primates (NHPs) comparing the most common clinical adjuvant aluminum hydroxide (alum) with saponin/monophosphoryl lipid A nanoparticles (SMNP), an immune-stimulating complex-like adjuvant. SMNP elicited substantially stronger humoral immune responses than alum, including 7-fold higher peak antigen-specific germinal center B-cell responses, 18-fold higher autologous neutralizing antibody titers, and higher levels of antigen-specific plasma and memory B cells. Positron emission tomography and computed tomography imaging in live NHPs showed that, unlike alum, SMNP promoted rapid antigen accumulation in both proximal and distal lymph nodes (LNs). SMNP also induced strong type I interferon transcriptional signatures, expansion of innate immune cells, and increased antigen-presenting cell activation in LNs. These findings indicate that SMNP promotes multiple facets of the early immune response relevant for enhanced immunity to vaccination.

Single and Combinatorial Effects of Metformin and Thymoquinone in Diffuse Large B Cell Lymphoma Cells.

Diffuse large B cell lymphoma (DLBCL) is classified into Germinal Center B-cell (GCB) and activated B-cell (ABC) subgroups originating from different stages of lymphoid differentiation. Cell of origin dictates the behavior and therapeutic response of DLBCL. This study aimed to evaluate single and combinatorial effects of metformin and thymoquinone (TQ) in two DLBCL cell lines belonging to GCB and ABC subtypes. Metformin and TQ caused dose-dependent responses in both ABC and GCB DLBCL subtypes. Metformin had a greater impact on the ABC subtype while TQ demonstrated more pronounced effects on the GCB subtype. Synergistic effects were observed in the DHL4 (GCB subtype) but not in the HBL1 (ABC subtype) cell line. This is the first study to compare the effects of metformin and TQ in ABC versus GCB subtype of DLBCL. It brings valuable results that could be utilized in further research aimed at reshaping treatments for subtype-specific lymphomas.

Multilevel Analysis of MYC and BCL2 Aberrations in Diffuse Large B-Cell Lymphoma: Identifying a High-Risk Patient Subgroup Across Cell-of-Origin Using Targeted Sequencing.

Diffuse large B-cell lymphoma (DLBCL) exhibits striking clinical and biological heterogeneity. Recent studies have identified new subgroups within germinal center B-cell like (GCB) DLBCL, associated with inferior prognosis, irrespective of MYC and BCL2 translocations. We explored the existence of such a DLBCL high-risk subgroup, based on multilevel aberrations, especially focusing on MYC and BCL2. Tissue samples from 111 DLBCL patients were sequenced with a 90-gene lymphoma panel, followed by integrative analyses combining sequencing data, immunohistochemistry, fluorescent insitu hybridization, and clinical data. We identified a high-risk subgroup in DLBCL defined by: dual immunohistochemical MYC and BCL2 expression (DEL), concurrent MYC and BCL2 translocations (DHL-BCL2), mutations in MYC, CXCR4, or both, and/or BCL2 amplification. The high-risk subgroup constituted 41% of the cohort and included DHL-BCL2, DEL, a GCB subgroup likely representing the recently described GCB subgroups, and a subset of non-GCB patients. In multivariate analysis, high-risk features provided independent predictive value from age and IPI. The 5-year overall survival was 36% in high-risk patients, compared to 76% in non-high-risk patients. We identified a distinct high-risk DLBCL subgroup, characterized by MYC and BCL2 aberrations, beyond conventional DHL-BCL2 and DEL, and irrespective of cell-of-origin, thereby expanding the poor-prognosis group.

Biomarker potential of nuclear Nrf2 activation in the ABC subtype of diffuse large B‑cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell lymphoma characterized by distinct subtypes and heterogeneous treatment outcomes. Oxidative stress and the dysregulation of related regulatory genes are prevalent in DLBCL, prompting an investigation into the nuclear factor erythroid 2-related factor 2 (Nrf2)-kelch-like ECH-associated protein 1 (Keap1) signaling pathway and associated genes. The present study assessed pathological specimens and clinical data from 43 newly diagnosed patients with DLBCL, comparing the associations and correlations between the expression of Nrf2, Keap1, microtubule-associated protein 1 light chain 3β (LC3B) and nitrotyrosine and the activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of DLBCL using immunohistochemistry and digital image analysis software. Nuclear Nrf2 activation was observed in 33.3% of patients with DLBCL ABC, demonstrating a higher prevalence of hepatitis B surface antigen positivity, calcium ions and significant body weight loss (P<0.05). Total Nrf2 expression was associated with the DLBCL GCB subtype and inversely correlated with Keap1 expression in the DLBCL ABC subtype. Furthermore, a positive correlation was demonstrated between Nrf2 and LC3, indicating that total Nrf2 is inhibited by Keap1 and regulates LC3 expression. The ABC subtype was also associated with lower white blood cell counts and more frequent chemotherapy courses than the GCB subtype. These findings suggest that nuclear Nrf2 could be a biomarker for DLBCL clinical diagnosis.

CNS relapse in high-grade B-cell lymphoma with MYC and BCL2 rearrangements and dark-zone signature–expressing DLBCL

AbstractHigh-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2), or “double-hit lymphoma,” has been associated with a high risk of central nervous system (CNS) relapse. However, historic estimates are impacted by selection bias. We report CNS relapse rates associated with HGBCL-DH-BCL2 from a population-based cohort with complete fluorescence in situ hybridization testing, as well as diffuse large B-cell lymphoma morphology (DLBCL) tumors expressing the dark-zone gene expression signature (DZsig), which was originally derived from HGBCL-DH-BCL2. The 2-year CNS relapse risk in HGBCL-DH-BCL2 was 6.8%. CNS relapses were early, predominantly leptomeningeal (73%), and co-occurred with systemic relapse (64%). High-risk CNS International Prognostic Index (CNS-IPI) and concordant bone marrow involvement were associated with an elevated CNS relapse risk in HGBCL-DH-BCL2. The “refined cell-of-origin” classification assigned 20% of DLBCL morphology tumors with germinal center B-cell–like phenotype (GCB-DLBCL) into a distinct subgroup based on DZsig expression (DZsig+). CNS relapse risk in DZsig+ (2 year: 6.4%) was independent of HGBCL-DH-BCL2 status and was further stratified by the CNS-IPI. CNS relapse in DZsig-negative GCB-DLBCL was rare (2-year risk, 1.4%; P = .04 vs DZsig+) and exclusively parenchymal. Altogether, the CNS relapse risk in HGBCL-DH-BCL2 is lower than previously reported, and DZsig refines risk stratification in GCB-DLBCL.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

CD30 influences germinal center B-cell dynamics and the expansion of IgG1-switched B cells

Initially, identified as a Hodgkin lymphoma marker, CD30 was subsequently detected on a subset of human B cells within and around germinal centers (GCs). While CD30 expression is typically restricted to a few B cells, expansion of CD30-expressing B cells occurs in certain immune disorders and during viral infections. The role of CD30 in B cells remains largely unclear. To address this gap in knowledge, we established a conditional CD30-knockin mouse strain. In these mice, B-cell-specific CD30 expression led to a normal B-cell phenotype in young mice, but most aged mice exhibited significant expansion of B cells, T cells and myeloid cells and increased percentages of GC B cells and IgG1-switched cells. This may be driven by the expansion of CD4+ senescence-associated T cells and T follicular helper cells, which partially express CD30-L (CD153) and may stimulate CD30-expressing B cells. Inducing CD30 expression in antigen-activated B cells accelerates the GC reaction and augments plasma cell differentiation, possibly through the posttranscriptional upregulation of CXCR4. Furthermore, CD30 expression in GC B cells promoted the expansion of IgG1-switched cells, which displayed either a GC or memory-like B-cell phenotype, with abnormally high IgG1 levels compared with those in controls. These findings shed light on the role of CD30 signaling in GC B cells and suggest that elevated CD30+ B-cell numbers lead to pathological lymphocyte activation and proliferation.

Regulation of BCR-dependent germinal center B-cell formation by HGAL and insight into its emerging myeloid ortholog, C1ORF150.

The specificity of cytokine and immunoreceptor signaling frequently depends upon receptor recruitment of select adaptor proteins and specifically engaged effectors. This review focuses on the orthologous adaptor proteins, HGAL and C1ORF150, and aims to provide insight into their respective modulation of lymphoid and myeloid cell signaling, formation, and function. HGAL acts predominantly within germinal center B cells as an important BCR signal transducer. Effects on BCR signalosome assembly involve HGAL's localization to the plasma membrane via its lipidation, initial interactions with SYK, the pY-phosphorylation of HGAL including its recruitment of GRB2, and HGAL engagement of PDZ-RhoGEF and RhoA signaling. At ligated BCRs, this includes HGAL(-GRB2) stimulation of SYK kinase, attenuation of calcium flux-dependent and NF-κB expression, promotion of cSMAC formation, and cytoskeletal remodeling associated with HGAL-attenuated cell migration. HGAL and partnered effectors also impact on DLBCL pathogenesis, and studies are summarized on HGAL's actions (using DLBCL and Burkitt lymphoma B cells) including cell migration effects, HGAL modulation of cytoskeletal components, and insightful HGAL transgenic mouse and xenograft models. For C1ORF150, its HGAL-homologous subdomains are considered, together with studies that demonstrate C1OR150's FcϵRI- and KIT-mediated expression and phosphorylation in primary human mast cells. Intriguingly, recent GWAS studies have identified a C1ORF150 in-frame splice variant that is strongly associated with urticaria. Candidate mechanisms via which the encoded "C1ORF150-Δexon2" isoform affects mast cell degranulation are considered, including FcϵR1 and/or KIT receptor connections, and candidate "myristoylation switch" mechanisms.

Utility of MEF2B Immunohistochemistry in Distinguishing Follicular Lymphoma and Marginal Zone Lymphoma in Diagnostically Challenging Cases

Abstract Introduction/Objective Distinguishing follicular lymphoma (FL) from marginal zone lymphoma (MZL) can be challenging, as the morphology and immunophenotype can overlap particularly in CD10-negative cases or those with marginal zone differentiation (MZD). MEF2B (Myocyte enhancer binding factor B) is a transcription factor that controls BCL6 expression in germinal center (GC) B-cells. Previous studies report MEF2B to have a high sensitivity and specificity for FL compared to other GC markers. We investigated the expression of MEF2B in low grade B cell lymphomas (LGBCL) to evaluate its utility in classifying diagnostically challenging cases and FL with MZD. Methods/Case Report Our archives were searched from 2005-2023 to identify excisional biopsies of LGBCL with MZD, including (FL), splenic MZL (SMZL), MZL of mucosa-associated lymphoid tissue (MALT), nodal MZL (NMZL) and difficult-to-classify (DTC) cases with a differential diagnosis of MZL and FL. MEF2B immunohistochemistry was performed retrospectively (Atlas Antibodies). Two hematopathologists assigned a pre-and post-MEF2B diagnosis to DTC cases. Results (if a Case Study enter NA) 57 cases were included: 21 FL (12 with MZD), 11 SMZL, 13 MALT, 4 NMZL, and 8 DTC cases. 19/21 FL cases (90%) were positive for MEF2B, including 11/12 (92%) cases with MZD. However, MEF2B expression was restricted to the follicles and negative in MZD areas in 7/11 cases (64%), while 4 cases showed partial weak expression in the areas of MZD. 0/11 SMZL (0%), 0/13 MALT (0%), and 0/4 (0%) NMZL were positive for MEF2B. 6/8 DTC cases (75%) were positive for MEF2B. Within DTC cases, retrospective inclusion of MEF2B results helped to render a more definitive diagnosis in 5 cases (2 negative/3 positive). Conclusion MEF2B immunohistochemistry can aid in distinguishing FL from MZL in challenging cases. However, it is often negative in areas of FL with MZD so care must be taken on interpretation in small biopsies.

Clinicopathological analysis of primary central nervous system lymphoma in patients with or without HIV infection

The clinicopathological features of HIV-related primary central nervous system lymphoma (PCNSL) and immunocompetent primary central nervous system lymphoma (IC-PCNSL) were found to be distinct. Thirty-seven patients with HIV-related PCNSL and thirty patients with IC-PCNSL were included in our study. Hematoxylin & eosin (HE) staining, immunohistochemical detection using CD10, MUM1, CD20, Bcl-2, Bcl-6, p53, C-MYC, Ki67, methyltransferase like factor 3 (METTL3) antibodies and Epstein–Barr encoding region (EBER) in situ hybridization were performed. All of the patients were classified as the diffuse large B-cell lymphoma (DLBCL) histological type. Patients with HIV-related PCNSL were younger and more likely to be male, with elevated lactate dehydrogenase (LDH) and low sugar content in cerebrospinal fluid (CSF) compared to patients with IC-PCNSL.The positive rates of METTL3, Bcl-2, p53 and EBER were significantly higher in HIV-related PCNSL patients than in IC-PCNSL patients. Furthermore, we also found that the expression of METTL3 was lower in germinal centre B-cell (GCB)-like DLBCL (n = 7) than in non-GCB like DLBCL (n = 30) in HIV-related PCNSL (P = 0.030); however, in IC-PCNSL patients, the expression of METTL3 was not significantly different between GCB-like DLBCL and non-GCB-like DLBCL (P = 0.670). Although the manifestations are similar in PCNSL patients with and without HIV, HIV-related PCNSL differs from IC-PCNSL in terms of pathological characteristics including METTL3, Bcl-2, p53 and EBER. We therefore suggest that the pathogenesis of HIV-related PCNSL and IC-PCNSL may differ according to host immune status.

Follicular lymphoma comprises germinal center–like and memory-like molecular subtypes with prognostic significance

AbstractA robust prognostic and biological classification for newly diagnosed follicular lymphoma (FL) using molecular profiling remains challenging. FL tumors from patients treated in the RELEVANCE trial with rituximab-chemotherapy (R-chemo) or rituximab-lenalidomide (R2) were analyzed using RNA sequencing, DNA sequencing, immunohistochemistry (IHC), and/or fluorescence in situ hybridization. Unsupervised gene clustering identified 2 gene expression signatures (GSs) enriched in normal memory (MEM) B-cells and germinal center (GC) B-cell signals, respectively. These 2 GSs were combined into a 20-gene predictor (FL20) to classify patients into MEM-like (n = 160) or GC-like (n = 164) subtypes, which also displayed different mutational profiles. In the R-chemo arm, patients with MEM-like FL had significantly shorter progression-free survival (PFS) than patients with GC-like FL (hazard ratio [HR], 2.13; P = .0023). In the R2 arm, both subtypes had comparable PFS, demonstrating that R2 has a benefit over R-chemo for patients with MEM-like FL (HR, 0.54; P = .011). The prognostic value of FL20 was validated in an independent FL cohort with R-chemo treatment (GSE119214 [n = 137]). An IHC algorithm (FLcm) that used FOXP1, LMO2, CD22, and MUM1 antibodies was developed with significant prognostic correlation with FL20. These data indicate that FL tumors can be classified into MEM-like and GC-like subtypes that are biologically distinct and clinically different in their risk profile. The FLcm assay can be used in routine clinical practice to identify patients with MEM-like FL who might benefit from therapies other than R-chemo, such as the R2 combination. This trial was registered at www.clinicaltrials.gov as #NCT01476787 and #NCT01650701.

Germinal center B-cell subgroups in the tumor microenvironment cannot be overlooked: Their involvement in prognosis, immunotherapy response, and treatment resistance in head and neck squamous carcinoma

BackgroundMore than 60 % of patients with head and neck squamous carcinoma (HNSCC) are diagnosed at advanced stages and miss radical treatment. This has prompted the need to find new biomarkers to achieve early diagnosis and predict early recurrence and metastasis of tumors. MethodsSingle-cell RNA sequencing (scRNA-seq) data from HNSCC tissues and peripheral blood samples were obtained through the Gene Expression Omnibus (GEO) database (GSE164690) to characterize the B-cell subgroups, differentiation trajectories, and intercellular communication networks in HNSCC and to construct a prognostic model of the associated risks. In addition, this study analyzed the differences in clinical features, immune cell infiltration, functional enrichment, tumor mutational burden (TMB), and drug sensitivity between the high- and low-risk groups. ResultsUsing scRNA-seq of HNSCC, we classified B and plasma cells into a total of four subgroups: naive B cells (NBs), germinal center B cells (GCBs), memory B cells (MBs), and plasma cells (PCs). Pseudotemporal trajectory analysis revealed that NBs and GCBs were at the early stage of B cell differentiation, while MBs and PCs were at the end. Cellular communication revealed that GCBs acted on tumor cells through the CD99 and SEMA4 signaling pathways. The independent prognostic value, immune cell infiltration, TMB and drug sensitivity assays were validated for the MEF2B+ GCB score groups. ConclusionsWe identified GCBs as B cell-specific prognostic biomarkers for the first time. The MEF2B+ GCB score fills the research gap in the genetic prognostic prediction model of HNSCC and is expected to provide a theoretical basis for finding new therapeutic targets for HNSCC.

High-grade B-cell lymphoma, not otherwise specified: CNS involvement and outcomes in a multi-institutional series

AbstractLittle is known about the central nervous system (CNS) risk in high-grade B-cell lymphoma, not otherwise specified (HGBL NOS). Hence, we sought to describe the rates of baseline CNS involvement, risk of CNS recurrence after primary therapy, and management strategies in HGBL NOS. In this multicenter retrospective study, we included 160 adults with newly diagnosed HGBL NOS treated between 2016 and 2021 at 20 US institutions. Eleven patients (7%) had baseline CNS involvement at diagnosis (leptomeningeal = 6, parenchymal = 4, and both = 1). Baseline CNS involvement was significantly associated only with MYC rearrangement (OR = 3.5) and testicular (in men) or female pelvic (in women) involvement (OR = 8.1). There was no significant difference in survival outcomes between patients with HGBL NOS with (median PFS = 4 years) or without (median PFS = 2.4 years) baseline CNS involvement (P = 0.45). The cumulative incidence of CNS recurrence at 3 years was 11%. Patients with baseline CNS involvement were at the highest risk (48.5% vs 8% for those without baseline CNS involvement) and were excluded from the risk factors analysis for CNS recurrence. The risk for CNS recurrence was significantly associated with blood or bone marrow involvement, CD5 expression, non–germinal center B-cell subtype, and “dual-expresser lymphoma” phenotype, however, high CNS IPI was not. The prognosis of relapsed HGBL NOS was poor, regardless of whether recurrence was systemic or limited to the CNS, and with currently available salvage strategies, including autologous transplantation and chimeric antigen receptor T-cell modalities, almost all patients with CNS recurrence ultimately succumbed to their disease.

MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis

The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B-cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S)324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B-cell lymphomagenesis.