Microenvironment Of Diffuse Large B-cell Lymphoma Research Articles

Chidamide enhances T-cell-mediated anti-tumor immune function by inhibiting NOTCH1/NFATC1 signaling pathway in ABC-type diffuse large B-cell lymphoma

Chidamide (CS055/HBI-8000, tucidinostat) has shown promising effects in the clinical treatment of various hematologic tumors. Diffuse large B-cell lymphoma (DLBCL) has shown highly heterogeneous biological characteristics. There are complex mechanisms of the role of chidamide in DLBCL for in-depth study. It is essential to probe further into the mechanism of drug-tumor interactions as a guide to clinical application and to understand the occurrence and progression of DLBCL. In vitro and in vivo models were utilized to determine the effects of chidamide on signaling pathways involved in the DLBCL tumor microenvironment. The experimental results show that chidamide inhibited the proliferation of DLBCL cell lines in a dose- and time-dependent manner, and down-regulated the expression of NOTCH1 and NFATC1 in DLBCL cells as well as decreased the concentration of IL-10 in the supernatant. In addition, chidamide significantly lowered the expression of PD1 or TIM3 on CD4+T cells and CD8+T cells and elevated the levels of IL-2, IFN-γ, and TNF-α in the serum of animal models, which augmented the function of circulating T cells and tumor-infiltrating T cells and ultimately significantly repressed the growth of tumors. These findings prove that chidamide can effectively inhibit the cell activity of DLBCL cell lines by inhibiting the activation of NOTCH1 and NFATC1 signaling pathways. It can also improve the abnormal DLBCL microenvironment in which immune escape occurs, and inhibit immune escape. This study provides a new therapeutic idea for the exploration of individualized precision therapy for patients with malignant lymphoma.

Lymphoma-Derived IL-10 Is a Key Immunomodulatory Factor at the Tumor Microenvironment of Activated B-Cell Diffuse Large B-Cell Lymphoma and Influences In Vivo Responses to Immunotherapy

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease to R-CHOP immunochemotherapy. These higher risk patients often exhibit gene expression profiles that resemble a late germinal center-derived activated B-cell (ABC) phenotype, where diverse specific mutations in genes along BCR and TLR signaling pathways converge into constitutive activation of the NF-κB pathway. Identification of factors that contribute to the survival of malignant ABC-DLBCL cells is crucial for designing specific interventions. In this setting, previous in vitro studies in DLBCL cell lines have evidenced that IL-10 is a direct target of NF-κB signaling that sustains lymphoma cell survival by an auto-stimulatory loop via STAT3 signaling. However, the action of IL-10 could be more complex within the lymphoma microenvironment, as IL-10 can have both stimulatory and inhibitory effects depending on the cellular source of IL-10, the timing of its secretion, and the type of immune cells that receives signals via IL-10 receptor (IL-10R). To explore in vivo the relevance of lymphoma-derived IL-10, we have generated a novel quintuple transgenic mouse model that we call pBIC10, after crossing our previous ABC-DLBCL-like pBIC mice carrying constitutive NF-κB signaling (Pascual et al., Blood 2019) to mice with a floxed Il10 gene for conditional knock-out of IL-10 specifically in malignant ABC-DLBCL cells. As expected, increased intracellular IL-10 signaling was detected in primary murine DLBCL cells from pBIC mice but not in IL-10 deficient pBIC10 mice, which promoted lymphoma cell survival ex vivo as revealed by perturbation studies of the IL-10/JAK1/STAT3 autocrine loop with blocking monoclonal antibodies or selected inhibitors. However, in vivo genetic deletion of lymphoma-derived IL-10 did not offer improved overall survival but, on the contrary, notably accelerated DLBCL progression in pBIC10 mice (Figure 1A, left). A comprehensive integration of multiparametric flow cytometry and transcriptomic analyses using bulk and single-cell RNA-seq of lymphoma cells and the tumor microenvironment coupled with BCR- and TCR-seq, identified lymphoma-derived IL-10 as a key immunomodulator of the DLBCL microenvironment, including unexpected protective paracrine functions in the progression of DLBCL. By comparing IL-10-proficient (pBIC) and -deficient (pBIC10) murine DLBCL models, we observed a multifaceted role of IL-10 produced by DLBCL cells, enabling immune-effector processes (facilitating immune chemotaxis, antigen presentation, and IFNγ responses) that yielded higher percentages of long-lived stem-like CD8 +TCF-1 +PD-1 lo/int T cells and dendritic cells, while restraining PD-1/LAG3-driven T-cell exhaustion and reducing immunosuppression by CD4 +CD25 +FOXP3 + regulatory T (Treg) cells. Thus, depletion of lymphoma-derived IL-10 decreased T-cell cytotoxicity and increased Treg-driven immunosuppression, which collectively accelerated lymphoma development. Consequently, T-cell targeted immune-checkpoint therapy (with anti-PD-1 antibodies) was effective only in IL-10-proficient DLBCL but not in IL-10-deficient pBIC10 mice (Figure 1A, right). Further comparative studies between pBIC and pBIC10 mice showed that tumor vascularization and expression of B-cell calcium channels, which have been previously associated with sensitivity to rituximab, were counteracted by lymphoma-derived IL-10. Accordingly, anti-CD20-based targeting of lymphoma cells (with rituximab surrogate antibodies) showed best in vivo responses in IL-10-deficient DLBCL mice with respect to pBIC mice (Figure 1A, right). Consistent with this preclinical data, analyses of transcriptional IL-10-associated hallmarks to stratify DLBCL patients (Schmitz et al., NEJM 2018) identified better clinical responses to R-CHOP in patients with pBIC10-like DLBCL phenotype (Figure 1B). Collectively, our study provides new functional and mechanistic insights into the role of lymphoma-derived IL-10 in the pathogenesis of DLBCL and the restraint of an immunosuppressed tumor microenvironment, which holds predictive potential as a biomarker for immunotherapy responses in DLBCL.

The Characterization and Prognostic Value of Tumor Immune Microenvironment in Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is featured by high heterogeneity, especially in clinical outcome. Tumor microenvironment (TME) represents a complex ecosystem around tumor cells, which has been found to be associated with the prognosis of DLBCL patients. However, the overall profile of TME immune components, perception of its correlation with tumor development and molecular mechanism are still insufficient in DLBCL. Here, we aimed to investigate novel prognostic scoring system and biomarkers based on TME immune components, and explore their underlying molecular mechanism in the development of DLBCL. Methods: Microarray transcriptomic data and corresponding clinical information of DLBCL patients (n=946) from GSE23501, GSE53786, GSE10846, GSE136971, GSE57611 and GSE32918 in GEO database were analyzed. The immune composition was calculated by CIBERSORT. Immunohistochemistry (IHC) was used to assess the expression level of CD2 in DLBCL tissues. Seurat in R was employed for conducting single-cell analysis. The study was performed with the approval of the Medical Ethical Committee of Shandong Provincial Hospital, and all samples were collected with informed consents. Results: To assess the immune cell composition of TME in DLBCL patients, we conducted a comprehensive analysis of the relative proportions of 22 distinct immune cell types within DLBCL TME. Subsequently, the prognostic role of TME immune components in DLBCL patients was probed, and a novel TME immune score based on the proportion of 22 immune cells was established. Furthermore, to evaluate the association between TME immune score and clinical prognosis in DLBCL patients, we performed K-M analysis and 3,5,10-year ROC analysis, which revealed the high prognostic efficacy of TME immune score. Higher TME immune scores were demonstrated to be significantly associated with worse prognosis, which was subsequently validated in an external cohort containing 230 DLBCL patients. Survival data of quartile subgroups also unveiled the prognostic value of TME immune score in DLBCL patients ( Fig. 1A). We further explored the correlation between TME immune score and clinical pathological features in DLBCL patients. Higher TME immune scores were observed to be correlated with more advanced Ann Arbor stage, non-germinal center B-cell-like subtype, lower expression levels of immune checkpoint molecules (CTLA-4, PD-1, PD-L2), decreased ESTIMATE stromal and immune scores, as well as higher tumor purity, indicating that the TME immune score may enable the prediction of treatment response to immune checkpoint inhibitors. The potential molecular mechanism of TME modulation in DLBCL development was further explored. Results of GO and KEGG enrichment illuminated that differential expression genes (DEGs) in two groups were significantly enriched in the anti-tumor immunity related processes and pathways, especially T-cell receptor activation. Subsequently, 6 hub genes (CCL5, CD2, CD3E, MMP9, CCR5, GATA3) in protein-protein interaction network constructed by DEGs were identified, whose expression levels were found to be associated with prognosis in DLBCL patients. Moreover, IHC was used to investigate the relevance of CD2 protein expression with prognosis in DLBCL patients. The expression of CD2 protein was found to be associated with poor prognosis in DLBCL patients ( Fig. 1A). To further investigate the regulatory function of CD2 in the immune microenvironment of DLBCL, we conducted single-cell analysis based on three DLBCL patients, and revealed that CD2 was prominently expressed in T cell subpopulation and was correlated with different differentiation trajectory of T cells in DLBCL. Subsequent differential gene analysis revealed that DEGs between state 1 and state 2, 3 were significantly enriched in KEGG pathways including antigen presentation and Th cell differentiation ( Fig. 1B), suggesting the role of CD2 in T cell activation and differentiation. Conclusions: Taken together, we established a novel TME immune score which could serve as a prognostic indicator in DLBCL patients. More importantly, we identified CD2 as a prognostic biomarker in DLBCL patients and unveiled its association with different T-cell activation states. Our study might provide a novel prognostic stratification strategy and promising biomarkers, which could facilitate the individualized management in DLBCL patients.

Role and application of chemokine CXCL13 in central nervous system lymphoma.

Chemokine ligand 13 (CXCL13) and its chemokine receptor 5 (CXCR5) both play significant roles in the tumor microenvironment (TME). CXCL13 in cerebrospinal fluid (CSF) has recently been found to have significant diagnostic and prognostic value in primary and secondary central nervous system (CNS) diffuse large B-cell lymphoma (DLBCL), and the CXCL13-CXCR5 axis has been shown to play an important chemotactic role in the TME of CNS-DLBCL. In this review, we first describe the clinical value of CXCL13 in CSF as a prognostic and diagnostic biomarker for CNS-DLBCL. In addition, this review also discusses the specific mechanisms associated with the CXCL13-CXCR5 axis in tumor immunity of primary diffuse large B cell lymphoma of the central nervous system (PCNS-DLBCL) by reviewing the specific mechanisms of this axis in the immune microenvironment of DLBCL and CNS inflammation, as well as the prospects for the use of CXCL13-CXCR5 axis in immunotherapy in PCNS-DLBCL.

Blockade of PD-1 and LAG-3 expression on CD8+ T cells promotes the tumoricidal effects of CD8+ T cells.

The diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed. LAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry. According to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SU-DHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis. High LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL.

A unique expression pattern of LAG3 distinct from that of other immune checkpoints in diffuse large B-cell lymphoma.

Although some patients with diffuse large B-cell lymphoma (DLBCL) show a response to immunotherapy, there are still many who do not respond. This suggests that various immune checkpoints are complicatedly intertwined in the composition of the tumor microenvironment of DLBCL. To comprehensively understand the expression of various immune checkpoint genes in DLBCL, we performed NanoString assay in 98 patients to investigate 579 genes. In addition, we performed immunohistochemistry for LAG-3 and PD-L1 to compare the results with expression in NanoString assay. As a result of hierarchical clustering of NanoString assay, 98 DLBCLs were classified into three tumor immune microenvironment clusters. Most immune checkpoint genes showed the highest expression in cluster A and the lowest in cluster C. However, the expression of LAG3 was the highest in cluster C and the lowest in cluster A, showing an expression pattern opposite to that of other immune checkpoint genes. In Cluster A, the expression of genes related to T-cell activity such as CD8A and GZMB was increased. In Cluster C, the expression of genes related to major histocompatibility complex molecules was the highest. Immunohistochemical stains showed modest agreement with the NanoString results but did not help clustering. Our results show that the unique expression pattern of LAG3 in DLBCL contrasts with that of other immune checkpoints. We suggest that the combination of anti-PD-1/PD-L1 and anti-LAG-3 blockades in the immunotherapy of DLBCL patients can have a synergistic effect, improving the immunotherapy efficacy and outcome in DLBCL patients.

Machine Learning‐Based Stem Cell‐Like Phenotype Identification and Novel Risk Stratification in Diffuse Large B‐Cell Lymphoma: Multi‐Omics Data from Multicenter Studies

Background: Previous researches have mainly focused on the discussion of whether cancer stem cells exist in diffuse large B-cell lymphoma (DLBCL). However, some subgroups with dismal prognosis that exhibit stem cell-like characteristics have been overlooked. Methods: Using an innovative one-class logistic regression (OCLR) machine learning algorithm, we calculated stemness indices and systematically assessed their ability to reflect the oncogenic dedifferentiation-like characteristics of DLBCL. Next, we identified signatures associated with oncogenic dedifferentiation-like features and prognosis using LASSO and SVM-RFE algorithms and developed a proportional hazards model (Riskscore) for DLBCL through Cox regression. Then we stratified the risk of DLBCL (n = 2133) and evaluated the prognostic value of the risk model across known clinical and molecular subgroups. We further compared the characteristics of high- and low-risk DLBCL with Burkitt lymphoma. Finally, we investigated the mechanisms of poor prognosis in high-risk DLBCL using transcriptomics, genomics (n = 576) and single-cell RNA sequencing (n = 19) data, as well as cell experiments and internal validation cohorts. Results: In this study, we identified and validated a DLBCL subgroup (25.6% of DLBCL) with stem cell-like characteristics and dismal prognosis. This high-risk group was defined as polyamine metabolism-cold immune tumor with upregulated polyamine metabolic key enzyme (ODC1) and desert-like immune infiltration, and had a poor prognosis with lower 3-year OS rate (54.3% vs. 83.6%, p < 0.0001) and PFS rate (42.8% vs. 75.2%, p < 0.0001) compared to the low-risk group. We found that some patients with MYC rearrangement, double-hit, double-expresser, or complete remission may have either favorable or poor prognosis, which can be accurately identified by our risk stratification model. Additionally, the high-risk group exhibited malignant proliferative phenotypes similar to Burkitt lymphoma. Genomic analysis revealed widespread copy number losses in the chemokine and interferon coding regions 8p23.1 and 9p21.3 in the high-risk group. Bulk and single-cell transcriptome analysis indicated that the upregulation of ODC1 might mediate the cold immune microenvironment of DLBCL, and knocking down ODC1 effectively inhibited DLBCL cell proliferation. Promisingly, our model effectively identified patients who were insensitive to immunotherapy (CAR-T and immune checkpoint antibodies). The research was funded by: This work was supported by grants from the National Natural Science Foundation of China (grant no. 81873450, 82170181), Beijing Hospitals Authority Youth Programme (code: QML20200201), and Beijing Natural Science Foundation (No. 7222027) to Liang Wang. Keywords: Bioinformatics, Computational and Systems Biology, Microenvironment, Risk Models No conflicts of interests pertinent to the abstract.

Diffuse large B-cell lymphoma microenvironment displays a predominant macrophage infiltrate marked by a strong inflammatory signature.

Inflammasomes are cytosolic signaling hubs that promote the inflammatory response (i.e. an immune reaction to counteract threats in physiological conditions). Their potential role in lymphomagenesis remains to be elucidated. Depending on the context, innate immune cells, such as macrophages, may induce inflammation that contributes to the anti-tumor function; however, if uncontrolled, inflammation can promote cancer development. Here, we exploited bioinformatic tools, TCGA data, and tumor tissue samples from patients with diffuse large B-cell lymphoma (DLBCL), one of the most frequent non-Hodgkin lymphomas of B-cell origin, to investigate the distribution of the different immune cell subpopulations in DLBCL samples in order to characterize the immune landscape of their microenvironment. We found a clear prominence of macrophages in the DLBCL microenvironment. Particularly, the proportions of resting M0 and pro-inflammatory M1 macrophages were higher in DLBCL than spleen samples (controls). As each inflammasome has unique sensor activation and platform assembly mechanisms, we examined the expression of a large panel of inflammasome actors. We found that inflammasome components, cytokines and Toll-like receptors were upregulated in DLBCL samples, particularly in M0 and M1 macrophages, compared with controls. Moreover, their expression level was positively correlated with that of CD68 (a pan-macrophage marker). We confirmed the positive correlation between CD68 and IRF8 expression at the protein level in DLBCL tissue samples, where we observed increased infiltration of CD68- and IRF8-positive cells compared with normal lymph nodes. Altogether, our results highlight the inflammatory status of the DLBCL microenvironment orchestrated by macrophages. More work is needed to understand the complexity and potential therapeutic implications of inflammasomes in DLBCL.

The Immunology of DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy and is the most common type of malignant lymphoid neoplasm. While some DLBCLs exhibit strong cell-autonomous survival and proliferation activity, others depend on interactions with non-malignant cells for their survival and proliferation. Recent next-generation sequencing studies have linked these interactions with the molecular classification of DLBCL. For example, germinal center B-cell-like DLBCL tends to show strong associations with follicular T cells and epigenetic regulation of immune recognition molecules, whereas activated B-cell-like DLBCL shows frequent genetic aberrations affecting the class I major histocompatibility complex. Single-cell technologies have also provided detailed information about cell-cell interactions and the cell composition of the microenvironment of DLBCL. Aging-related immunological deterioration, i.e., immunosenescence, also plays an important role in DLBCL pathogenesis, especially in Epstein-Barr virus-positive DLBCL. Moreover, DLBCL in "immune-privileged sites"-where multiple immune-modulating mechanisms exist-shows unique biological features, including frequent down-regulation of immune recognition molecules and an immune-tolerogenic tumor microenvironment. These advances in understanding the immunology of DLBCL may contribute to the development of novel therapies targeting immune systems.

Prognostic Value of PIK3R4 Expression and Its Correlation with Immune Cell Infiltration in the Diffuse Large B-cell Lymphoma Microenvironment.

Background: As a regulatory unit of class III phosphoinositide 3-kinase (PI3K), PIK3R4 is an important molecule involved in several malignant tumours, but the role and molecular mechanism of PIK3R4 in diffuse large B-cell lymphoma (DLBCL) is still unclear. Methods: Multiple bioinformatics analyses were used to investigate the role and potential mechanisms of PIK3R4 in DLBCL. Quantitative real-time polymerase chain reaction (qRT‒PCR) was performed to determine the expression of PIK3R4 in 80 DLBCL patients, and the survival time of DLBCL patients grouped according to PIK3R4 mRNA expression was compared. Results: PIK3R4 is up-regulated in several malignant tumours, including DLBCL. Bioinformatics analyses revealed that PIK3R4 exhibits prognostic value in DLBCL patients, and the upregulation of this gene in DLBCL samples was subsequently validated. In the functional category, GO analysis revealed that PIK3R4-related genes are enriched in ribosomal RNA metabolic process, the DNA damage response, mitochondrial gene expression, and nucleoside metabolic process. KEGG pathway analysis showed the enrichment of PIK3R4-related genes in the ribosome, oxidative phosphorylation, proteasome, and cellular senescence pathways. More importantly, the expression of PIK3R4 in DLBCL was correlated with the immune cell content in the cancer microenvironment, CD8(+) T-cell and neutrophil infiltration and the levels of several immune checkpoint molecules, including BTN3A2, BTN3A1, PRF1, CXCL9, PDCD1, and TIGIT. Conclusion: Our study demonstrated that PIK3R4, as a novel immune microenvironment-related gene, may represent an important diagnostic, prognostic, or therapeutic biomarker in DLBCL patients.

Blocking the CD47-Sirpa Axis Enhances Tafasitamab-Mediated Phagocytosis

Introduction Around 40% of diffuse large B cell lymphoma (DLBCL) patients have refractory disease or experience relapse, thus highlighting the need for novel, more effective therapies (Coiffier et al). The immunotherapy Tafasitamab (MOR208) is an Fc-modified, humanized, monoclonal anti-CD19 antibody approved in combination with lenalidomide as a second line of therapy for R/R DLBCL. Tafasitamab mediates its therapeutic effect directly or via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Macrophages represent a key component of the DLBCL microenvironment and are critical effectors of antibody therapies (Uchida et al). However, ADCP is reportedly counteracted by increased expression of the immune checkpoint molecule CD47 on lymphoma targets (Chao et al). In addition, the inhibitory receptor of CD47, signal regulatory protein alpha (SIRPα) is increased on macrophages from patients with follicular lymphoma who relapse or progress after frontline lenalidomide and rituximab (Marques-Piubelli et al). Aim of the study The current study investigates the impact of the CD47-SIRPα axis on tafasitamab-mediated phagocytosis and the potential of concomitant treatment with an anti-CD47 blocking antibody to improve its efficacy. Results We show that both, CD47 and its inhibitory receptor SIRPa were elevated in lymph node biopsies from DLBCL patients. CD47 expression was evaluated in 6 DLBCL patients and 5 healthy controls and SIRPα was evaluated in 9 patients and 7 controls. In fact, increased expression of SIRPa on macrophages correlated with decreased ADCP activity of tafasitamab, and CRISPR-mediated CD47 overexpression on lymphoma targets impaired tafasitamab-mediated phagocytosis, in vitro. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination therapy. Importantly, the combination enhanced tafasitamab-mediated phagocytosis of primary DLBCL cells using primary, patient-derived lymphoma-associated macrophages (LAMs) as effectors, in an autologous setting. Conclusion Our results show increased expression of CD47-SIRPa axis in primary DLBCL samples and indicate that CD47 blockade can enhance the phagocytic potential of tumor-targeting immunotherapies such as tafasitamab. Our findings suggest that there is merit in exploring the clinical utility of combining tafasitamab with an anti-CD47 blocking antibody in lymphoma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrative analysis of a necroptosis-related gene signature of clinical value and heterogeneity in diffuse large B cell lymphoma.

Background: Diffuse large B-cell lymphoma (DLBCL), which is considered to be the most common subtype of lymphoma, is an aggressive tumor. Necroptosis, a novel type of programmed cell death, plays a bidirectional role in tumors and participates in the tumor microenvironment to influence tumor development. Targeting necroptosis is an intriguing direction, whereas its role in DLBCL needs to be further discussed. Methods: We obtained 17 DLBCL-associated necroptosis-related genes by univariate cox regression screening. We clustered in GSE31312 depending on their expressions of these 17 genes and analyzed the differences in clinical characteristics between different clusters. To investigate the differences in prognosis across distinct clusters, the Kaplan-Meier method was utilized. The variations in the tumor immune microenvironment (TME) between distinct necroptosis-related clusters were investigated via “ESTIMATE”, “Cibersort” and single-sample geneset enrichment analysis (ssGSEA). Finally, we constructed a 6-gene prognostic model by lasso-cox regression and subsequently integrated clinical features to construct a prognostic nomogram. Results: Our analysis indicated stable but distinct mechanism of action of necroptosis in DLBCL. Based on necroptosis-related genes and cluster-associated genes, we identified three groups of patients with significant differences in prognosis, TME, and chemotherapy drug sensitivity. Analysis of immune infiltration in the TME showed that cluster 1, which displayed the best prognosis, was significantly infiltrated by natural killer T cells, dendritic cells, CD8+ T cells, and M1 macrophages. Cluster 3 presented M2 macrophage infiltration and the worst prognosis. Importantly, the prognostic model successfully differentiated high-risk from low-risk patients, and could forecast the survival of DLBCL patients. And the constructed nomogram demonstrated a remarkable capacity to forecast the survival time of DLBCL patients after incorporating predictive clinical characteristics. Conclusion: The different patterns of necroptosis explain its role in regulating the immune microenvironment of DLBCL and the response to R-CHOP treatment. Systematic assessment of necroptosis patterns in patients with DLBCL will help us understand the characteristics of tumor microenvironment cell infiltration and aid in the development of tailored therapy regimens.

Clinical Impact of Immune Cells and Their Spatial Interactions in Diffuse Large B-Cell Lymphoma Microenvironment.

Tumor-infiltrating immune cells have prognostic significance and are attractive therapeutic targets. Yet, the clinical significance of their spatial organization and phenotype in diffuse large B-cell lymphoma (DLBCL) is unclear. We characterized T cells, macrophages, and their spatial interactions by multiplex IHC (mIHC) in 178 patients with DLBCL and correlated the data with patient demographics and survival. We validated the findings on gene expression data from two external DLBCL cohorts comprising 633 patients. Macrophage and T-cell contents divided the samples into T cell-inflamed (60%) and noninflamed (40%) subgroups. The T cell-inflamed lymphoma microenvironment (LME) was also rich in other immune cells, defining immune hot phenotype, which did not as such correlate with outcome. However, when we divided the patients according to T-cell and macrophage contents, LME characterized by high T-cell/low macrophage content or a corresponding gene signature was associated with superior survival [5-year overall survival (OS): 92.3% vs. 74.4%, P = 0.036; 5-year progression-free survival (PFS): 92.6% vs. 69.8%, P = 0.012]. High proportion of PD-L1- and TIM3-expressing CD163- macrophages in the T cell-inflamed LME defined a group of patients with poor outcome [OS: HR = 3.22, 95% confidence interval (CI), 1.63-6.37, Padj = 0.011; PFS: HR = 2.76, 95% CI, 1.44-5.28, Padj = 0.016]. Furthermore, PD-L1 and PD-1 were enriched on macrophages interacting with T cells. Our data demonstrate that the interplay between macrophages and T cells in the DLBCL LME is immune checkpoint dependent and clinically meaningful.

Impact of Qi-Invigorating Traditional Chinese Medicines on Diffuse Large B Cell Lymphoma Based on Network Pharmacology and Experimental Validation.

Background: It has been verified that deficiency of Qi, a fundamental substance supporting daily activities according to the Traditional Chinese Medicine theory, is an important symptom of cancer. Qi-invigorating herbs can inhibit cancer development through promoting apoptosis and improving cancer microenvironment. In this study, we explored the potential mechanisms of Qi-invigorating herbs in diffuse large B cell lymphoma (DLBCL) through network pharmacology and in vitro experiment. Methods: Active ingredients of Qi-invigorating herbs were predicted from the Traditional Chinese Medicine Systems Pharmacology Database. Potential targets were obtained via the SwissTargetPrediction and STITCH databases. Target genes of DLBCL were obtained through the PubMed, the gene-disease associations and the Malacards databases. Overlapping genes between DLBCL and each Qi-invigorating herb were collected. Hub genes were subsequently screened via Cytoscape. The Gene Ontology and pathway enrichment analyses were performed using the DAVID database. Molecular docking was performed among active ingredients and hub genes. Hub genes linked with survival and tumor microenvironment were analyzed through the GEPIA 2.0 and TIMER 2.0 databases, respectively. Additionally, in vitro experiment was performed to verify the roles of common hub genes. Results: Through data mining, 14, 4, 22, 22, 35, 2, 36 genes were filtered as targets of Ginseng Radix et Rhizoma, Panacis Quinquefolii Radix, Codonopsis Radix, Pseudostellariae Radix, Astragali Radix, Dioscoreae Rhizoma, Glycyrrhizae Radix et Rhizoma for DLBCL treatment, respectively. Then besides Panacis Quinquefolii Radix and Dioscoreae Rhizoma, 1,14, 10, 14,13 hub genes were selected, respectively. Molecular docking studies indicated that active ingredients could stably bind to the pockets of hub proteins. CASP3, CDK1, AKT1 and MAPK3 were predicted as common hub genes. However, through experimental verification, only CASP3 was considered as the common target of Qi-invigorating herbs on DLBCL apoptosis. Furthermore, the TIMER2.0 database showed that Qi-invigorating herbs might act on DLBCL microenvironment through their target genes. Tumor-associated neutrophils may be main target cells of DLBCL treated by Qi-invigorating herbs. Conclusion: Our results support the effects of Qi-invigorating herbs on DLBCL. Hub genes and immune infiltrating cells provided the molecular basis for each Qi-invigorating herb acting on DLBCL.

High PTX3 expression is associated with a poor prognosis in diffuse large B-cell lymphoma.

Tumor‐associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B‐cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein–Barr virus‐encoded small RNA (EBER)‐positive and five high‐grade B‐cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B‐cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan‐macrophages), CD16 (M1‐like), CD163, pentraxin 3 (PTX3), and interleukin (IL)‐10‐positive macrophages (M2c‐like) and low infiltration of FOXP3‐positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell‐like DLBCL was associated with high CD16, CD163, PTX3, and IL‐10, and EBER‐positive DLBCL with high CD163 and PTX3. Programmed cell death‐ligand 1 positively correlated with CD16, CD163, IL‐10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll‐like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America (GSE10846, R‐CHOP‐like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c‐like immune regulatory macrophages and low infiltration of FOXP3‐positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.

The landscape of tumor cell states and ecosystems in diffuse large B cell lymphoma.

Biological heterogeneity in diffuse large B cell lymphoma (DLBCL) is partly driven by cell-of-origin subtypes and associated genomic lesions, but also by diverse cell types and cell states in the tumor microenvironment (TME). However, dissecting these cell states and their clinical relevance at scale remains challenging. Here, we implemented EcoTyper, a machine-learning framework integrating transcriptome deconvolution and single-cell RNA sequencing, to characterize clinically relevant DLBCL cell states and ecosystems. Using this approach, we identified five cell states of malignant B cells that vary in prognostic associations and differentiation status. We also identified striking variation in cell states for 12 other lineages comprising the TME and forming cell state interactions in stereotyped ecosystems. While cell-of-origin subtypes have distinct TME composition, DLBCL ecosystems capture clinical heterogeneity within existing subtypes and extend beyond cell-of-origin and genotypic classes. These results resolve the DLBCL microenvironment at systems-level resolution and identify opportunities for therapeutic targeting (https://ecotyper.stanford.edu/lymphoma).

The microenvironment of DLBCL is characterized by noncanonical macrophages recruited by tumor-derived CCL5

AbstractTissue invasion by tumor cells induces a host inflammatory response that variably impacts tumorigenesis. This has been well documented for tumor-associated macrophages (TAMs) that could play a pro/M2- or an anti/M1-tumoral function. TAMs frequently infiltrate diffuse large B-cell lymphoma (DLBCL), an aggressive neoplasm arising from germinal center–experienced B cells. However, the pathway leading to the presence of TAMs in DLBCL remains unknown, and their impact is unclear. Here, we show that some DLBCL tumor cells expressed the chemokine CCL5, enabling the differential recruitment of blood monocytes through their expression of CCR1 and CCR5. CCL5 expression by DLBCL was not related to molecular subtypes, and healthy tonsillar B cells did not produce this chemokine, implying a posttransformation event. A single-cell analysis revealed that most DLBCL TAMs had a noncanonical gene signature with the concomitant expression of M1 and M2 genes. The presence of noncanonical TAMs may explain the lack of impact of macrophages on DLBCL development reported in some survival studies.

Characteristics of PD-L1 expression in tumor cells and tumor microenvironment of DLBCL with MYD88 L265P mutation

Objective: To study the effect of MYD88 L265P mutation on the expression of PD-L1 in tumor cells and tumor microenvironment in diffuse large B-cell lymphoma (DLBCL), and to provide theoretical basis for immunotherapy for patients. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect the frequency of MYD88 L265P mutation in 72 cases of DLBCL diagnosed by pathologists in Cancer Hospital of Chinese Academy of Medical Sciences from August 2008 to May 2010. Expression of PD-L1 in tumor cells and tumor microenvironment in all samples was evaluated using PD-L1 (22C3) and PD-L1 (SP142) with Ventana automatic immunohistochemical (IHC) platform. The relationship between MYD88 L265P mutation and the expression of PD-L1 in DLBCL tumor cells and tumor microenvironment was assessed. Results: Of the 72 cases of DLBCL, MYD88 L265P mutation was detected in 15 (20.8%) cases. Nine cases with JAK2 amplification were excluded, and the remaining 63 cases of DLBCL were divided into MYD88 L265P mutant group (n=14) and MYD88 L265P wild-type group (n=49). IHC results showed that among the 14 cases of MYD88 L265P mutant groups, PD-L1 (22C3) was positive in 7 cases (7/14) of tumor cells and PD-L1 (SP142) was positive in 4 cases (4/14) of tumor microenvironment. Among the 49 cases of MYD88 L265P wild-type group, 9 cases (18.4%) were positive for PD-L1 (22C3) in tumor cells, and 38 cases (77.6%) were positive for PD-L1(SP142) in tumor microenvironment. In addition, among the 16 cases with PD-L1(22C3) expression in tumor cells, only 2 of the 7 cases with MYD88 L265P mutation were positive for PD-L1 (SP142) in tumor microenvironment. All 9 cases with wild-type MYD88 L265P were positive for PD-L1 (SP142) in tumor microenvironment. Statistical analysis showed that the expression level of PD-L1 (22C3) in tumor cells in the MYD88 L265P mutant group was significantly higher than that in the MYD88 L265P wild-type group (P=0.017). The expression level of PD-L1 (SP142) in tumor microenvironment in the MYD88 L265P mutant group was significantly lower than that in the MYD88 L265P wild-type group (P=0.001). Conclusions: MYD88 L265P mutation may play an important role in the regulation of PD-L1 expression in DLBCL tumor cells and tumor microenvironment. Further studies will provide a theoretical basis for immunotherapy of DLBCL patients with MYD88 L265P mutation.

In This Issue

In This Issue

Clinical and Biological Subtypes of B-cell Lymphoma Revealed by Microenvironmental Signatures.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease. Transcriptomic and genetic characterization of DLBCL has increased the understanding of its intrinsic pathogenesis and provided potential therapeutic targets. However, the role of the microenvironment in DLBCL biology remains less understood. Here, we performed a transcriptomic analysis of the microenvironment of 4,655 DLBCLs from multiple independent cohorts and described four major lymphoma microenvironment categories that associate with distinct biological aberrations and clinical behavior. We also found evidence of genetic and epigenetic mechanisms deployed by cancer cells to evade microenvironmental constraints of lymphoma growth, supporting the rationale for implementing DNA hypomethylating agents in selected patients with DLBCL. In addition, our work uncovered new therapeutic vulnerabilities in the biochemical composition of the extracellular matrix that were exploited to decrease DLBCL proliferation in preclinical models. This novel classification provides a road map for the biological characterization and therapeutic exploitation of the DLBCL microenvironment. SIGNIFICANCE: In a translational relevant transcriptomic-based classification, we characterized the microenvironment as a critical component of the B-cell lymphoma biology and associated it with the DLBCL clinical behavior establishing a novel opportunity for targeting therapies.This article is highlighted in the In This Issue feature, p. 1307.