Cystatin SN (CST1) Is a Poor Independent Prognostic Biomarker for Gastrointestinal Diffuse Large B-Cell Lymphoma.

BACKGROUND Gastrointestinal diffuse large B-cell lymphoma (GI-DLBCL) is the most common histological subtype of extra-nodal DLBCL, but the risk factors, prognostic biomarkers, histopathological classifications, and treatment strategies have not had significant progress. Emerging evidence shows that cystatin SN (CST1) is involved in tumor progression in several cancer types, but its role in GI-DLBCL has not been revealed. MATERIAL AND METHODS We established a cohort consisting of 84 patients with GI-DLBCL who underwent surgical resection. The expression of CST1 in the cohort was investigated by immunohistochemistry, which divided the patients into subgroups with low or high expression of CST1. Moreover, the CST1 expression in GI-DLBCL tissues or adjacent GI tissues were compared with RT-qPCR. The correlation between CST1 expression and clinicopathological factors was analyzed with the chi-square test. The prognostic significance of CST1 was estimated by univariate and multivariate analysis, and statistical significance was analyzed with the log-rank test. RESULTS CST1 was aberrantly upregulated in GI-DLBCL tissues compared with in non-tumor GI tissues. High expression of CST1 indicated poor prognosis of GI-DLBCL (P=0.012), and CST1 can be regarded as an independent prognostic biomarker of GI-DLBCL (hazard ratio=3.07). In our study, serum lactate dehydrogenase (P=0.002), performance status (P=0.003), Lugano stage (P=0.002), and International Prognostic Index (P=0.001) were also prognostic factors of GI-DLBCL. CONCLUSIONS CST1 is an independent prognostic biomarker of GI-DLBCL, indicating unfavorable prognosis. Our results suggested that CST1 detection can be a promising method to stratify high-risk patients and guide individual treatment.

[Effects of polyphyllin â ¦ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells].

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

N-acetyltransferase 10 facilitates tumorigenesis of diffuse large B-cell lymphoma by regulating AMPK/mTOR signalling through N4-acetylcytidine modification of SLC30A9.

BACKGROUND: Accumulating studies suggested that posttranscriptional modifications exert a vital role in the tumorigenesis of diffuse large B-cell lymphoma (DLBCL). N4-acetylcytidine (ac4C) modification, catalyzed by the N-acetyltransferase 10 (NAT10), was a novel type of chemical modification that improves translation efficiency and mRNA stability. METHODS: GEO databases and clinical samples were used to explore the expression and clinical value of NAT10 in DLBCL. CRISPER/Cas9-mediated knockout of NAT10 was performed to determine the biological functions of NAT10 in DLBCL. RNA sequencing, acetylated RNA immunoprecipitation sequencing (acRIP-seq), LC-MS/MS, RNA immunoprecipitation (RIP)-qPCR and RNA stability assays were performed to explore the mechanism by which NAT10 contributed to DLBCL progression. RESULTS: Here, we demonstrated that NAT10-mediated ac4C modification regulated the occurrence and progression of DLBCL. Dysregulated N-acetyltransferases expression was found in DLBCL samples. High expression of NAT10 was associated with poor prognosis of DLBCL patients. Deletion of NAT10 expression inhibited cell proliferation and induced G0/G1 phase arrest. Furthermore, knockout of NAT10 increased the sensitivity of DLBCL cells to ibrutinib. AcRIP-seq identified solute carrier family 30 member 9 (SLC30A9) as a downstream target of NAT10 in DLBCL. NAT10 regulated the mRNA stability of SLC30A9 in an ac4C-dependent manner. Genetic silencing of SLC30A9 suppressed DLBCL cell growth via regulating the activation of AMP-activated protein kinase (AMPK) pathway. CONCLUSION: Collectively, these findings highlighted the essential role of ac4C RNA modification mediated by NAT10 in DLBCL, and provided insights into novel epigenetic-based therapeutic strategies.

Palmitic acid reduces the methylation of the FOXO1 promoter to suppress the development of diffuse large B-cell lymphoma via modulating the miR-429/DNMT3A axis.

Diffuse large B-cell lymphoma (DLBCL) is characterized by significant treatment resistance. Palmitic acid (PA) has shown promising antitumor properties. This study aims to elucidate the molecular mechanisms by which PA influences DLBCL progression. We quantified the expression levels of microRNAs (miRNAs), Forkhead box protein O1 (FOXO1), and DNA methyltransferase 3A (DNMT3A) in both untreated and PA-treated DLBCL tumors and cell lines. Assessments were made of cell viability, apoptosis, and autophagy-related protein expression following PA administration. Interaction analyses among miR-429, DNMT3A, and FOXO1 were conducted using luciferase reporter assays and methylation-specific (MSP) Polymerase chain reaction (PCR). After transfecting the miR-429 inhibitor, negative control (NC) inhibitor, shRNA against DNMT3A (sh-DNMT3A), shRNA negative control (sh-NC), overexpression vector for DNMT3A (oe-DNMT3A), or overexpression negative control (oe-NC), we evaluated the effects of miR-429 and DNMT3A on cell viability, mortality, and autophagy-related protein expression in PA-treated DLBCL cell lines. The efficacy of PA was also tested in vivo using DLBCL tumor-bearing mouse models. MiR-429 and FOXO1 expression levels were downregulated, whereas DNMT3A was upregulated in DLBCL compared to the control group. PA treatment was associated with enhanced autophagy, mediated by the upregulation of miR-429 and downregulation of DNMT3A. The luciferase reporter assay and MSP confirmed that miR-429 directly inhibits DNMT3A, thereby reducing FOXO1 methylation. Subsequent experiments demonstrated that PA promotes autophagy and inhibits DLBCL progression by upregulating miR-429 and modulating the DNMT3A/FOXO1 axis. In vivo PA significantly reduced the growth of xenografted tumors through its regulatory impact on the miR-429/DNMT3A/FOXO1 axis. Palmitic acid may modulate autophagy and inhibit DLBCL progression by targeting the miR-429/DNMT3A/FOXO1 signaling pathway, suggesting a novel therapeutic target for DLBCL management.

Cholesterol efflux from C1QB-expressing macrophages is associated with resistance to chimeric antigen receptor T cell therapy in primary refractory diffuse large B cell lymphoma.

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in early trials for relapsed/refractory diffuse large B cell lymphoma (DLBCL). However, its efficacy in treating primary refractory DLBCL has not been comprehensively investigated, and the underlying resistance mechanisms remain unclear. Here, we report the outcomes of a phase I, open-label, single-arm clinical trial of relmacabtagene autoleucel (relma-cel), a CD19-targeted CAR-T cell product, with safety and efficacy as primary endpoints. Among the 12 enrolled patients, 8 experienced grade 4 hematologic toxicity of treatment-emergent adverse event. No grade ≥3 cytokine release syndrome or neurotoxicity occurred. Single-cell RNA sequencing revealed an increase proportion of C1QB-expressing macrophages in patients with progressive disease before CAR-T cell therapy. Cholesterol efflux from M2 macrophages was found to inhibit CAR-T cells cytotoxicity by inducing an immunosuppressive state in CD8+ T cells, leading to their exhaustion. Possible interactions between macrophages and CD8+ T cells, mediating lipid metabolism (AFR1-FAS), immune checkpoint activation, and T cell exhaustion (LGALS9-HAVCR2, CD86-CTLA4, and NECTIN2-TIGIT) were enhanced during disease progression. These findings suggest that cholesterol efflux from macrophages may trigger CD8+ T cell exhaustion, providing a rationale for metabolic reprogramming to counteract CAR-T treatment failure. Chinadrugtrials.org.cn identifier: CTR20200376.

[Diagnosis and differential diagnosis of large B-cell lymphoma with IRF4 rearrangement].

Objective: To analyze the clinicopathological features and differential diagnosis of large B-cell lymphoma with IRF4 rearrangement, aiming enhance its recognition and prevent misdiagnosis. Methods: The clinicopathological features, immunophenotype, and fluorescence in situ hybridization (FISH) results of six cases diagnosed with IRF4 rearrangement-positive B-cell lymphoma at the Affiliated Hospital of Xuzhou Medical University from 2015 to 2023 were retrospectively analyzed. Additionally, a comprehensive review of the literature was conducted. Results: Six patients with IRF4 rearrangement-positive large B-cell lymphoma were included. Patients 1 to 5 included three males and two females with a median age of 19 years ranging from 11 to 34 years. Four patients presented with head and neck lesions, while the other one had a breast nodule; all were in clinical Ann Arbor stages I to Ⅱ. Morphologically, entirely diffuse pattern was present in two cases, purely follicular pattern in one case, and diffuse and follicular patterns in other two cases. The tumor cells, predominantly centroblasts mixed with some irregular centrocytes, were of medium to large size, with a starry sky appearance observed in two cases. Immunophenotyping revealed all cases were positive for bcl-6 and MUM1, with a Ki-67 index ranging from 70% to 90%, and CD10 was positive in two cases. IRF4 rearrangement was confirmed in all cases by FISH analysis, with dual IRF4/bcl-6 rearrangements identified in two cases, leading to a diagnosis of LBCL-IRF4. Case 6, a 39-year-old female with a tonsillar mass and classified as clinical Ann Arbor stage Ⅳ, displayed predominantly diffuse large B-cell lymphoma (DLBCL) morphology with 20% high-grade follicular lymphoma characteristics. Immunohistochemistry showed negative CD10 and positive bcl-6/MUM1, with a Ki-67 index of approximately 80%. Triple rearrangements of IRF4/bcl-2/bcl-6 were identified by FISH, leading to a diagnosis of DLBCL with 20% follicular lymphoma (FL). All six patients achieved complete remission after treatment, with no progression or relapse during a follow-up period of 31-100 months. Conclusions: Large B-cell lymphoma with IRF4 rearrangement is a rare entity with pathological features that overlap with those of FL and DLBCL. While IRF4 rearrangement is necessary for diagnosing LBCL-IRF4, it is not specific and requires differentiation from other aggressive B-cell lymphomas with IRF4 rearrangement.

C-MYC-activated lncRNA SNHG20 accelerates the proliferation of diffuse large B cell lymphoma via USP14-mediated deubiquitination of ß-catenin.

BACKGROUND: Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has been recognized as a critical lncRNA in multiple human cancers. However, the role of SNHG20 and its underlying mechanism in DLBCL are still unclear. METHODS: The expression levels of SNHG20, c-MYC, ß-catenin, and ubiquitin-specific peptidase 14 (USP14) were measured by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR) and immunoblotting. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) incorporation, and flow cytometry assays were used to assess the proliferation and apoptosis of DLBCL cells. The transcriptional regulation of SNHG20 by c-MYC was confirmed by a luciferase reporter assay and RNA immunoprecipitation. The interaction between USP14 and ß-catenin was demonstrated using coimmunoprecipitation. A subcutaneous xenograft model was constructed to determine the role of SNHG20 in vivo. RESULTS: In the present study, we found that SNHG20 expression was upregulated in DLBCL cell lines and tissues compared to their normal counterparts. SNHG20 knockdown prominently reduced the proliferation and induced the apoptosis of U2932 and OCI-LY3 cells. However, SNHG20 overexpression increased the proliferation and apoptosis resistance of DLBCL cells. Mechanistically, the expression of SNHG20 was positively regulated by c-MYC in DLBCL cells. C-MYC directly bound to the promoter of SNHG20 to activate its transcription. SNHG20 was expressed mainly in the cytosol in DLBCL cells. SNHG20 silencing did not impact USP14 expression but markedly decreased the level of ß-catenin, the substrate of USP14, in DLBCL cells. USP14 overexpression increased the ß-catenin level, and this increase was attenuated by SNHG20 knockdown. Treatment with the proteasome inhibitor MG132 abolished SNHG20 knockdown-induced ß-catenin downregulation. Moreover, SNHG20 silencing reduced the half-life but increased the ubiquitination of ß-catenin in DLBCL cells. SNHG20 knockdown weakened the interaction between both endogenous and exogenous USP14 and ß-catenin. In turn, SNHG20 overexpression increased the c-MYC level, and this increase was attenuated by ß-catenin knockdown. Importantly, ß-catenin knockdown attenuated the SNHG20-mediated increase in DLBCL cell proliferation in vitro and tumour growth in vivo. CONCLUSIONS: Taken together, our results suggested that c-MYC-activated SNHG20 accelerated the proliferation and increased the apoptosis resistance of DLBCL cells via USP14-mediated deubiquitination of ß-catenin. The c-MYC/SNHG20 positive feedback loop may be a new target for anti-DLBCL treatment.

Molecular Changes in Immunological Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells in Lymphoid Neoplasia.

Immune system and bone marrow stromal cells play an important role in maintaining normal hematopoiesis. Lymphoid neoplasia disturbs not only development of immune cells, but other immune response mechanisms as well. Multipotent mesenchymal stromal cells (MSCs) of the bone marrow are involved in immune response regulation through both intercellular interactions and secretion of various cytokines. In hematological malignancies, the bone marrow stromal microenvironment, including MSCs, is altered. Aim of this study was to describe the differences of MSCs' immunological function in the patients with acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In ALL, malignant cells arise from the early precursor cells localized in bone marrow, while in DLBCL they arise from more differentiated B-cells. In this study, only the DLBCL patients without bone marrow involvement were included. Growth parameters, surface marker expression, genes of interest expression, and secretion pattern of bone marrow MSCs from the patients with ALL and DLBCL at the onset of the disease and in remission were studied. MSCs from the healthy donors of corresponding ages were used as controls. It has been shown that concentration of MSCs in the bone marrow of the patients with ALL is reduced at the onset of the disease and is restored upon reaching remission; in the patients with DLBCL this parameter does not change. Proliferative capacity of MSCs did not change in the patients with ALL; however, the cells of the DLBCL patients both at the onset and in remission proliferated significantly faster than those from the donors. Expression of the membrane surface markers and expression of the genes important for differentiation, immunological status maintenance, and cytokine secretion differed significantly in the MSCs of the patients from those of the healthy donors and depended on nosology of the disease. Secretomes of the MSCs varied greatly; a number of proteins associated with immune response regulation, differentiation, and maintenance of hematopoietic stem cells were depleted in the secretomes of the cells from the patients. Lymphoid neoplasia leads to dramatic changes in the functional immunological status of MSCs.

Advances in proteomics in diffuse large B­cell lymphoma (Review).

Diffuse large B­cell lymphoma (DLBCL) is the most common pathological type of non­Hodgkin's lymphoma. Although the development of monoclonal antibodies, small­molecule­targeted drugs and novel chemotherapeutic agents, and the increased use of immunotherapy have markedly improved the outcomes of DLBCL, ~40% of patients cannot be cured following the use of standardized first­line treatment. In addition, the specific mechanisms of drug resistance and potential factors associated with a poor prognosis in these patients remain unclear. Proteomics research is used to determine potential associations between changes in DLBCL protein expression levels and different stages of disease occurrence and development. Proteomics may aid in the identification of novel molecular mechanisms and drug resistance mechanisms, through identifying multiple associated proteins and monitoring changes in expression levels. Thus, proteomics research may exhibit potential in the development of therapeutic targets and in improving prognostic evaluation in patients with DLBCL. The present study aimed to review the use of proteomic methods for the investigation of DLBCL, including the mechanisms underlying disease progression and drug resistance in DLBCL, and the function of the tumor microenvironment in lymphoma growth. The present review also demonstrated the potential of proteomic­guided therapeutic strategies for DLBCL and discussed the synergistic benefits of using proteomic methods in DLBCL research.

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas.

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.

Metabolic pathway-based subtyping reveals distinct microenvironmental states associated with diffuse large B-cell lymphoma outcomes.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease that requires personalized clinical treatment. Assigning patients to different risk categories and cytogenetic abnormality and genetic mutation groups has been widely applied for prognostic stratification of DLBCL. Increasing evidence has demonstrated that dysregulated metabolic processes contribute to the initiation and progression of DLBCL. Metabolic competition within the tumor microenvironment is also known to influence immune cell metabolism. However, metabolism- and immune-related stratification has not been established. Here, 1660 genes involved in 84 metabolic pathways were selected and tested to establish metabolic clusters (MECs) of DLBCL. MECs established based on independent lymphoma datasets distinguished different survival outcomes. The CIBERSORT algorithm and EcoTyper were applied to quantify the relative abundance of immune cell types and identify variation in cell states for 13 lineages comprising the tumor micro environment among different MECs, respectively. Functional characterization showed that MECs were an indicator of the immune microenvironment and correlated with distinctive mutational characteristics and oncogenic signaling pathways. The novel immune-related MECs exhibited promising clinical prognostic value and potential for informing DLBCL treatment decisions.

Arsenic disulfide promoted the demethylation of PTPL1 in diffuse large B cell lymphoma cells.

Background: Promoter hypermethylation of the tumor suppressor gene is one of the well-studied causes of cancer development. The drugs that reverse the process by driving demethylation could be a candidate for anticancer therapy. This study was designed to investigate the effects of arsenic disulfide on PTPL1 methylation in diffuse large B cell lymphoma (DLBCL). Methods: We knocked down the expression of PTPL1 in two DLBCL cell lines (i.e., DB and SU-DHL-4 cells) using siRNA. Then the DLBCL proliferation was determined in the presence of PTPL1 knockdown. The methylation of PTPL1 in DLBCL cells was analyzed by methylation specific PCR (MSPCR). The effect of arsenic disulfide on the PTPL1 methylation was determined in DLBCL cell lines in the presence of different concentrations of arsenic disulfide (5 µM, 10 µM and 20 µM), respectively. To investigate the potential mechanism on the arsenic disulfide-mediated methylation, the mRNA expression of DNMT1, DNMT3B and MBD2 was determined. Results: PTPL1 functioned as a tumor suppressor gene in DLBCL cells, which was featured by the fact that PTPL1 knockdown promoted the proliferation of DLBCL cells. PTPL1 was found hypermethylated in DLBCL cells. Arsenic disulfide promoted the PTPL1 demethylation in a dose-dependent manner, which was related to the inhibition of DNMTs and the increase of MBD2. Conclusion: Experimental evidence shows that PTPL1 functions as a tumor suppressor gene in DLBCL progression. PTPL1 hyper-methylation could be reversed by arsenic disulfide in a dose-dependent manner.

FAT1 inhibits the proliferation of DLBCL cells via increasing the m6A modification of YAP1 mRNA.

Emerging evidence shows that FAT atypical cadherin 1 (FAT1) mutations occur in lymphoma and are associated with poorer overall survival. Considering that diffuse large B cell lymphoma (DLBCL) is the category of lymphoma with the highest incidence rate, this study aims to explore the role of FAT1 in DLBCL. The findings demonstrate that FAT1 inhibits the proliferation of DLBCL cell lines by downregulating the expression of YAP1 rather than by altering its cellular localization. Mechanistic analysis via meRIP-qPCR/luciferase reporter assays showed that FAT1 increases the m6A modification of YAP1 mRNA 3'UTR and the subsequent binding of heterogeneous nuclear ribonucleoprotein D (HNRNPD) to the m6A modified YAP1 mRNA, thus decreasing the stability of YAP1 mRNA. Furthermore, FAT1 increases YAP1 mRNA 3'UTR m6A modification by decreasing the activity of the TGFß-Smad2/3 pathway and the subsequent expression of ALKBH5, which is regulated at the transcriptional level by Smad2/3. Collectively, these results reveal that FAT1 inhibits the proliferation of DLBCL cells by increasing the m6A modification of the YAP1 mRNA 3'UTR via the TGFß-Smad2/3-ALKBH5 pathway. The findings of this study therefore indicate that FAT1 exerts anti-tumor effects in DLBCL and may represent a novel target in the treatment of this form of lymphoma.

The Impact of Tumor Hypoxia Modulation on sIL-2R Levels in Newly Diagnosed Diffuse Large B Cell Lymphoma (DLBCL) Patients Undergoing Chemotherapy: A Randomized Clinical Trial.

OBJECTIVE: Tumor hypoxia induces the production of Hypoxia-Inducible Factor (HIF)-1 alpha, which interacts with NF-kB, leading to cancer proliferation and metastasis. This study investigated the effect of tumor hypoxia modulation using carbogen (95% O2 and 5% CO2) and nicotinamide on reducing soluble interleukin-2 receptor (sIL-2R) levels in newly diagnosed DLBCL patients with tissue overexpression of HIF-1α ≥10%. MATERIAL AND METHODS: A prospective randomized controlled clinical trial was conducted at Dr. Kariadi Hospital in Semarang, Indonesia, from 2021 to 2022. Newly diagnosed DLBCL patients with tissue HIF-1α ≥10% were randomized into an intervention group (nicotinamide 2,000 mg + carbogen 10 liters/min during R-CHOP) and a control group (R-CHOP alone) for one cycle. sIL-2R levels were measured in the blood before and after intervention. RESULTS: The intervention group showed a significant reduction in sIL-2R levels after chemotherapy (p=0.026), with 85% of samples exhibiting a decrease. In contrast, only 45% of samples in the control group demonstrated a decrease in sIL-2R levels (p=0.184). The median sIL-2R level decreased from 139.50 pg/mL to 70.50 pg/mL in the intervention group, while the control group exhibited an increase from 182.50 pg/mL to 250.00 pg/mL following one cycle of chemotherapy. CONCLUSION: Tumor hypoxia modulation led to a significant decrease in serum sIL-2R levels, potentially through improvements in the crosstalk between hypoxia and inflammation pathways.

Hyper-N-glycosylated SEL1L3 as auto-antigenic B-cell receptor target of primary vitreoretinal lymphomas.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress into primary central nervous system lymphoma (PCNSL). To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed B-cell receptors (BCR) from PVRL patients and tested for binding against human auto-antigens. SEL1L3, a protein with multiple glycosylation sites, was identified as the BCR target in 3/20 PVRL cases. SEL1L3 induces proliferation and BCR pathway activation in aggressive lymphoma cell lines. Moreover, SEL1L3 conjugated to a toxin killed exclusively lymphoma cells with respective BCR-reactivity. Western Blot analysis indicates the occurrence of hyper-N-glycosylation of SEL1L3 at aa 527 in PVRL patients with SEL1L3-reactive BCRs. The BCR of a PVRL patient with serum antibodies against SEL1L3 was cloned from a vitreous body biopsy at diagnosis and of a systemic manifestation at relapse. VH4-04*07 was used in both lymphoma manifestations with highly conserved CDR3 regions. Both BCRs showed binding to SEL1L3, suggesting continued dependence of lymphoma cells on antigen stimulation. These results indicate an important role of antigenic stimulation by post-translationally modified auto-antigens in the genesis of PVRL. They also provide the basis for a new treatment approach targeting unique lymphoma BCRs with ultimate specificity.

Recombinant hirudin suppresses angiogenesis of diffuse large B-cell lymphoma through regulation of the PAR-1-VEGF.

Hirudin is one of the specific inhibitors of thrombin, which has been confirmed to have strong bioactivities, including inhibiting tumors. However, the function and mechanism of hirudin and protease-activated receptor 1 (PAR-1) in diffuse large B-cell lymphoma (DLBCL) have not been clear. Detecting the expression PAR-1 in DLBCL tissues and cells by RT-qPCR and IHC. Transfected sh-NC, sh-PAR-1, or pcDNA3.1-PAR-1 in DLBCL cells or processed DLBCL cells through added thrombin, Vorapaxar, Recombinant hirudin (RH), or Na2S2O4 and co-culture with EA.hy926. And built DLBCL mice observed tumor growth. Detecting the expression of related genes by RT-qPCR, Western blot, IHC, and immunofluorescence, measured the cellular hypoxia with Hypoxyprobe-1 Kit, and estimated the cell inflammatory factors, proliferation, migration, invasion, and apoptosis by ELISA, CCK-8, flow cytometry, wound-healing and Transwell. Co-immunoprecipitation and pull-down measurement were used to verify the relationship. PAR-1 was highly expressed in DLBCL tissues and cells, especially in SUDHL2. Na2S2O4 induced SUDHL2 hypoxia, and PAR-1 did not influence thrombin-activated hypoxia. PAR-1 could promote SUDHL2 proliferation, migration, and invasion, and it was unrelated to cellular hypoxia. PAR-1 promoted proliferation, migration, and angiogenesis of EA.hy926 or SUDHL2 through up-regulation vascular endothelial growth factor (VEGF). RH inhibited tumor growth, cell proliferation, and migration, promoted apoptosis of DLBCL, and inhibited angiogenesis by down-regulating PAR-1-VEGF. RH inhibits proliferation, migration, and angiogenesis of DLBCL cells by down-regulating PAR-1-VEGF.

[Expression of CD30 in Patients with Diffuse Large B-Cell Lymphoma and Clinical Significance].

OBJECTIVE: To investigate the expression and clinical significance of CD30 in patients with diffuse large B-cell lymphoma (DLBCL). METHODS: A retrospective analysis was conducted on 124 cases of primary DLBCL diagnosed at Changzhou Second People's Hospital Affiliated with Nanjing Medical University from January 2018 to July 2020. The expression of CD30 in patients with DLBCL was detected by immunohistochemical method, and the clinicopathological characteristics were analyzed and compared between CD30+ and CD30- groups. Kaplan-Meier analysis was used for survival analysis. The relationship between CD30 expression and clinical features and prognosis were analyzed. RESULTS: Among the 124 patients with DLBCL, 19 patients expressed CD30, and the positive rate is 15.32%. The clinico-pathological characteristics of CD30+ in patients with DLBCL were characterized by low age, more common in males, fewer extranodal lesions, lower international prognostic index (IPI), GCB type being more common in Hans subtype, and achieving better therapeutic effects (P < 0.05). However, there were no significant statistical differences in B-symptoms (P =0.323), Ann Arbor staging (P =0.197), Eastern Cooperative Oncology Group (ECOG) score (P =0.479), lactate dehydrogenase (LDH) (P =0.477), and the involvement of bone marrow (P =0.222). There were significant differences in OS and PFS between the CD30+ and CD30- groups (χ2=5.653, P =0.017; χ2＝4.109，P =0.043), the CD30+ group had a better prognosis than that of the CD30- group. The results of subgroup analysis showed that the CD30+ group in the IPI score=1-2, LDH elevated group had a better prognosis (P < 0.05). In the subgroups of Ann Arbor staging III-IV (P =0.055) and non GCB type (P =0.053), the CD30+ group had a good prognosis trend, but the difference was not statistically significant. The results of univariate analysis showed that the good prognosis of DLBCL patients was closely related to CD30+ expression, no B-symptoms, early Ann Arbor staging, low ECOG score, normal LDH, low IPI score, fewer extranodal involvement, and obtaining the best therapeutic effect as CR (all P <0.05). COX multivariate regression analysis showed that the presence of B-symptoms and achieving the best therapeutic effect as Non-CR were independent risk factors affecting the prognosis of DLBCL patients (P < 0.05). CONCLUSION: The CD30+ expression in DLBCL patients indicates a good prognosis and has certain diagnostic value in evaluating the prognosis of DLBCL patients.

TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma.

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

ALK-positive large B-cell lymphoma (ALK + LBCL) with aberrant CD3 expression.

ALK-positive ( +) large B cell lymphoma (ALK + LBCL) is a rare distinct subtype of diffuse large B cell lymphoma presenting with high stage and aggressive behavior. Although B cell markers such as CD20, CD19, and CD22 are generally negative, plasmacytic markers including CD138, CD38, and MUM1 are positive. T cell markers are negative with rare exceptions. We report an unusual case of ALK1 + LBCL in a 58-year-old man with partial expression of CD3 without other T cell antigen expression. The tissue was evaluated with flow cytometry, immunohistochemistry, fluorescent in situ hybridization, and gene rearrangement studies. Gene rearrangement studies for IGH and TCR gamma were performed. Flow cytometry did not demonstrate any abnormal lymphoid populations. Tissue sectioning shows a malignant plasmacytic large cell neoplasm which expresses CD45 but is negative for CD20, CD79a, and PAX5. Plasmacytic markers CD138 and MUM1 are positive with kappa light chain restriction. Strong granular cytoplasmic expression of ALK is present. FISH showing disrupted ALK supports the diagnosis while MYC, BCL6, and BCL2 are intact. Gene rearrangement studies show coexisting IGH and TCR gamma clones; however, the TCR peak was present within a polyclonal background suggesting the disputed cells are likely only a subset of the T cell population. ALK + LBCL can present with an ambiguous immunophenotype, which warrants the use of multiple B cell, T cell, and plasmacytic antibodies. CD3 expression in this entity is rare and of uncertain clinical significance, but warrants further study.

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.