Progression Of Diffuse Large B-cell Lymphoma Research Articles

Long Noncoding RNA SNHG12 Indicates the Prognosis and Accelerates Tumorigenesis of Diffuse Large B-Cell Lymphoma Through Sponging microR-195.

BackgroundSmall nucleolar RNA host gene 12 (SNHG12) expression is associated with multiple cancers, including renal cell carcinoma, prostate cancer, cervical cancer, nasopharyngeal carcinoma, colorectal cancer, and hepatocellular carcinoma. However, SNHG12 biological function is unclear in diffuse large B-cell lymphoma (DLBCL).MethodsSNHG12 expression and associated clinicopathological characteristics were evaluated in DLBCL tissues. CCK-8 and transwell assay were used to analyze the in vitro role of SNHG12 in DLBCL progression. The xenograft model was used to explore the in vivo role of SNHG12 in DLBCL growth. The physical interaction between SNHG12 and miR-195 was confirmed using bioinformatics analysis and a dual luciferase assay.ResultsSNHG12 expression was upregulated in DLBCL tissues and correlated with patients’ prognosis. SNHG12 downregulation inhibited cell growth, migration, and invasion of DLBCL cells in vitro, while its overexpression promoted these cellular processes. Moreover, SNHG12 knockdown repressed tumorigenesis of DLBCL cells in vivo. Further experiments demonstrated that miR-195 is a target of SNHG12 in DLBCL and that their expression negatively correlates in DLBCL. SNHG12 functioned as a competing endogenous RNA for miR-195 in DLBCL cells and miR-195 upregulation abolished the effects of SNHG12 on of DLBCL progression.ConclusionSNHG12 predicts poor clinical outcome and serves as a novel oncogene in DLBCL via miR-195 sponging. We also suggest that SNHG12 can be used as a potential therapeutic candidate for DLBCL patients.

Cancer Antigen 125 Serum Level in Head and Neck Diffuse Large B-Cell Lymphoma

Background : Diffuse Large B-Cell Lymphoma (DLBCL) is the most common subtype of the Non-Hodgkin Lymphoma (LNH). Until now, lactate dehydrogenase (LDH) is the only tumor marker to assess DLBCL progression; however, increased LDH is a relatively non-specific biomarker. Cancer antigen 125 (CA-125) serum level have been used as a tumor marker in ovarian cancer. The aim of this study was to explore the possible role of CA-125 serum level as a tumor marker in head and neck DLBCL. Methods : This was an observational descriptive study among consecutively sampled DLBCL patients. CA-125 serum level examination was carried out (ADVIA Centaur CA-125II) and described along with the clinical characteristics of DLBCL patients. Results : DLBCL was mostly observed in males (54.05%), most often in the 55-65 year age group (51.3%), with stage 1 DLBCL was the most prevalent (71.9%). The mean CA-125 serum level was 22.9 U/ml and increased in patients with advanced DLBCL. Conclusions : Increased CA-125 serum level in DLBCL, especially at advanced stages, suggests that CA-125 serum level may be of benefit as a tumor marker in the head and neck DLBCL. Further study is in need to explore the role of CA-125. 1. Kementerian Kesehatan Republik Indonesia. Data dan kondisi penyakit limfoma di Indonesia. InfoDatin Pusat Data dan Informasi Kementerian Kesehatan RI. 2015 [cited 2020 March 18] Available from: https://www.kemkes.go.id/resources/download/pusdatin/infodatin/infodatin-limfoma.pdf 2. Dwianingsih EK, Indrawati, Hardianti MS, Malueka RG , Iswar RR, Sutapa S, et al. Histopathological features of lymphoma in Yogyakarta, Indonesia. Asian Pac J Cancer Prev. 2016;17(9):4213–16. 3. Badan Penelitian dan Pengembangan Kesehatan Kementerian Kesehatan RI. Riset Kesehatan Dasar (RISKESDAS) 2013. Jakarta: Badan Penelitian dan Pengembangan Kesehatan Kementerian Kesehatan RI; 2013 4. Cheson BD, Fisher RI, Barrington SF, Cavalli F, Schwartz LH, Zucca E, et al. Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014; 32(27):3059–67. 5. Ziepert M, Hasenclever D, Kuhnt E, Glass B, Schmitz N, Pfreundschuh, et al. Standard International prognostic index remains a valid predictor of outcome for patients with aggressive CD20+B-cell lymphoma in the rituximab era. J Clin Oncol. 2010;28(14):2373-2380. 6. Gutiérrez A, Martínez-Serra J, Barceló B, Sampol A, Vinas L, Gonzales G, et al. Prognostic value of serum CA125 levels in diffuse large B-cell lymphoma: potential role of a new sex and age adjusted reference value. Int J Lab Hematol. 2010; 32(6 Pt 2): 582–9. 7. Dilek I, Ayakta H, Demir C, Meral C, Ozturk M. CA 125 levels in patients with Non-Hodgkin lymphoma and other hematologic malignancies. Clin Lab Haematol. 2005;27(1):51–5. 8. Memar B, Aledavood A, Shahidsales S, Ahadi M, Farzadnia, Raziee HR, et al. The prognostic role of tumor marker CA-125 in B-Cell non-Hodgkin’s Lymphoma. Iran J Cancer Prev. 2015;8(1):42–6. 9. Garg S, Goyal B. Evaluation of CA-125 as a prognostic marker in patients with Non-Hodgkin’s lymphoma. International Journal of Medical and Health Research. 2017;3(4):13–5. 10. Paoli CJ, Bach BA, Quach D, Tsai KT, Wong B, Kallich J. Performance status of real-world oncology patients before and after first course of chemotherapy. The Journal of Community and Supportive Oncology. 2014; 12(5):163–70. 11. Yadav C, Ahmad A, D’Souza B, Agarwal A, Nandini M, Prabhu KA, et al. Serum lactate dehydrogenase in Non-Hodgkin’s lymphoma: a prognostic indicator. Indian J Clin Biochem. 2016;31(2):240–2.

FOXM1-induced upregulation of lncRNA OR3A4 promotes the progression of diffuse large B-cell lymphoma via Wnt/β-catenin signaling pathway

Diffuse large B-cell lymphoma (DLBCL) is a leading cause of non-Hodgkin lymphomas. Existing researches have verified that long non-coding RNAs (lncRNAs) play crucial roles in the development of DLBCL, nevertheless, whether lncRNA OR3A4 has influences on the progression of DCBCL needs further exploration. In our study, it was revealed that the expression of OR3A4 was upregulated in DLBCL tumors and cell lines, and patients with high OR3A4 expression suffered from poor prognosis. Knockdown of OR3A4 suppressed cell proliferation and promoted cell apoptosis in DLBCL. Moreover, knockdown of OR3A4 inactivated Wnt/β-catenin signaling pathway, and Riluzole treatment could partially rescue the inhibitive effect of OR3A4 silencing on DLBCL cell proliferation. Furthermore, FOXM1 expression was discovered to be upregulated in DLBCL tissues, and it positively modulated the expression of OR3A4 at transcriptional leve. It was also revealed that FOXM1 knockdown inactivated Wnt/β-catenin signaling pathway. Finally, rescue assays confirmed that OR3A4 overexpression or the treatment of Riluzole could partially countervail the inhibitive effect of FOXM1 silencing on DLBCL progression. Taken together, FOXM1-induced upregulation of OR3A4 enhances the occurrence of DLBCL via Wnt/β-catenin signaling pathway.

A Novel Circulating 4-miRNA Prognostic Model in Diffuse Large B-Cell Lymphoma: Role of MDSCs and Th17 Cells on Lymphoma Progression

Background: MicroRNAs (miRNAs) have been emerging as prognostic biomarkers in diffuse large B-cell lymphoma (DLBCL). The aim of this study is to translate these findings into clinical prediction on lymphoma progression. Methods: Using genome-wide miRNA expression in serum samples of 20 patients with DLBCL at diagnosis, remission and at relapse, candidate miRNAs were identified to establish circulating prognostic model. DNA- and RNA-sequencing were performed on tumor samples to detect genetic mutations and signaling pathway dysregulation. Mechanism of action of miRNAs on oncogenic signaling and tumor microenvironment was analyzed in vitro and in vivo. Findings: A novel circulating 4-miRNA prognostic model (miR21, miR130b, miR155, and miR28) significantly predicted clinical outcome of DLBCL, independent on International Prognostic Index in the training cohort [n=279, HR=2.83 (95%CI 2.14-3.51), P<0.001] and in the validation cohort [n=225, NCT01852435, HR=2.71 (95%CI 1.91-3.50), P<0.001]. DNA-sequencing data showed no significant difference of tumor mutation burden between low-risk and high-risk groups of the 4-miRNA model. RNA-sequencing data showed that 4-miRNA model correlated with aberrant Ras signaling transduction. In B-lymphoma cells, modulation of the 4 miRNAs regulated IGF1 and JUN expression, resulting in alterations in MDSCs and Th17 cells. Clinically, comparing with low-risk group of the 4-miRNA model, high-risk group presented Ras signaling activation, increased MDSCs and Th17 cells, and immunosuppressive status in DLBCL. Interpretations: The easy-to-use circulating 4-miRNA prognostic model was effective to predict relapse and survival in DLBCL. Tumor microenvironment contributes to the underlying mechanism of the 4-miRNA model on DLBCL progression. Funding: This study was supported, in part, by research funding from the National Natural Science Foundation of China (81520108003, 81830007, 81670176, 81700191 and 81900193), Chang Jiang Scholars Program,Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support (20152206 and 20152208), Shanghai Sailing Program (19YF1430900), Clinical Research Plan of Shanghai hospital development center (SHDC, 16CR2017A), Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine (DLY201601), Multicenter Hematology-Oncology Programs Evaluation System (M-HOPES), Collaborative Innovation Center of Systems Biomedicine, and the Samuel Waxman Cancer Research Foundation. Declaration of Interests: All authors declare no potential conflict of interest. Ethics Approval Statement: The study was approved by the Institutional Review Boards of all M-HOPES centers with informed consent obtained from all patients in accordance with the Declaration of Helsinki.

Tumor ADAM10/ADAM17-Mediated PD-L1 Loss May Predict Poor Outcomes in Diffuse Large B Cell Lymphoma

Introduction Tumor surface matrix metalloproteases ADAM10 and ADAM17 are associated with poor outcomes in multiple malignancies. We previously showed that these proteases shed PD-L1 from lymphoma cell line Karpas-299 to produce soluble PD-L1 (sPD-L1) that induces CD8+ T cell apoptosis (1). While higher levels of soluble PD-L1 are associated with worse prognosis in patients with non-Hodgkin lymphoma treated with standard chemoimmunotherapy (cutoff of 1.52 ng/ml, 3-year OS of 76% versus 89%) (2), it is unknown whether the loss of PD-L1 on the surface of B cell lymphoma cells affects clinical outcomes. We hypothesized that tumors expressing low PD-L1 levels despite high PD-L1 mRNA levels would (1) produce higher ADAM10 or ADAM17 transcript levels and (2) predict poorer prognosis in diffuse large B cell lymphomas. Methods We queried the cancer genome atlas (TCGA) public database for cases of diffuse large B cell lymphoma (DLBCL) for which PD-L1 (CD274) protein levels are reported by reverse phase protein array (RPPA). We then queried TCGA for RNA-Seq data for CD274, ADAM10, and ADAM17 expressed as transcripts per million (FPKM). We re-normalized RPPA data and calculated a PD-L1 protein-to-mRNA ratio for each tumor sample. We then compared groups of high and low PD-L1 protein-to-mRNA ratios for ADAM10 and ADAM17 mRNA expression. We further compared groups of high and low PD-L1 protein-to-mRNA ratios for survival (adjusted for age at diagnosis). All statistical analyses were performed using R Statistical Software (R Foundation). Unpaired student's t-test assessed statistical differences in experimental groups except where otherwise indicated. Figures comprise box plots showing quartile values and individual data points. P&lt;0.05 was considered statistically significant. In figures, p values are denoted &lt;0.05 with *, &lt;0.01 with **, and &lt;0.001 with ***. Results Data were available for a total of 33 diffuse large B cell (DLBC) lymphomas. 18 of those samples were categorized as having low PD-L1 protein-to-mRNA ratios (cutoff 6.38e-6) with the remaining categorized as high ratio. DLBCL tumors demonstrating low PD-L1 protein-to-mRNA ratios expressed significantly higher ADAM10 (p=0.0237) and ADAM17 (p&lt;0.0001) transcripts than tumors expressing low PD-L1 protein-to-mRNA ratios (Figure 1). Furthermore, patients with tumors demonstrating low PD-L1 protein-to-mRNA ratios experienced significantly poorer survival according to likelihood ratio (6.6, p=0.01) and log rank (4.67, p=0.03) tests (Figure 2). Conclusions Our findings may explain a crucial phenomenon seen in diffuse large B cell lymphoma, namely that many purportedly PD-L1-positive tumors appear to express PD-L1 but do not respond to PD-(L)1 inhibitor therapy (3,4). This may be caused by the activity of ADAM10 and ADAM17 to cleave PD-L1, which we previously showed in lymphoma cell line Karpas-299 (1). This posits pre-treatment ADAM10/ADAM17 inhibition to "boost" PD-(L)1 inhibitor therapy in lymphoma. Our results contrast with previous findings that PD-L1-positive DLBCL progression free survival is inferior to PD-L1-negative DLBCL (5). This may be due to different methods of protein detection (immunohistochemistry versus RPPA) and the lack of localization of PD-L1 expression in tumor cells versus the microenvironment. Notably, this study is only correlative with a limited case number. Larger prospective studies will be needed to further elucidate this relationship. Acknowledgments The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

Long non-coding RNA SNHG16 promotes proliferation and inhibits apoptosis of diffuse large B-cell lymphoma cells by targeting miR-497-5p/PIM1 axis.

The aberrant expression and dysfunction of long non‐coding RNAs (lncRNAs) have been identified as critical factors governing the initiation and progression of different human cancers, including diffuse large B‐cell lymphoma (DLBCL). LncRNA small nucleolar RNA host gene 16 (SNHG16) has been recognized as a tumour‐promoting factor in various types of cancer. However, the biological role of SNHG16 and its underlying mechanism are still unknown in DLBCL. Here we disclosed that SNHG16 was overexpressed in DLBCL tissues and the derived cell lines. SNHG16 knockdown significantly suppressed cell proliferation and cell cycle progression, and it induced apoptosis of DLBCL cells in vitro. Furthermore, silencing of SNHG16 markedly repressed in vivo growth of OCI‐LY7 cells. Mechanistically, SNHG16 directly interacted with miR‐497‐5p by acting as a competing endogenous RNA (ceRNA) and inversely regulated the abundance of miR‐497‐5p in DLBCL cells. Moreover, the proto‐oncogene proviral integration site for Moloney murine leukaemia virus 1 (PIM1) was identified as a novel direct target of miR‐497‐5p. SNHG16 overexpression rescued miR‐497‐5p‐induced down‐regulation of PIM1 in DLBCL cells. Importantly, restoration of PIM1 expression reversed SNHG16 knockdown‐induced inhibition of proliferation, G0/G1 phase arrest and apoptosis of OCI‐LY7 cells. Our study suggests that the SNHG16/miR‐497‐5p/PIM1 axis may provide promising therapeutic targets for DLBCL progression.

Neutrophil Extracellular Traps Induced by IL8 Promote Diffuse Large B-cell Lymphoma Progression via the TLR9 Signaling.

More than 30% of patients with diffuse large B-cell lymphoma (DLBCL) experience treatment failure after first-line therapy. Neutrophil extracellular traps (NETs), a pathogen-trapping structure in tumor microenvironment, can promote the transition of autoimmunity to lymphomagenesis. Here, we investigate whether NETs play a novel role in DLBCL progression and its underlying mechanism.Experimental Design: NETs in DLBCL tumor samples and plasma were detected by immunofluorescence and ELISA, respectively. The correlation between NETs and clinical features were analyzed. The effects of NETs on cellular proliferation and migration and mechanisms were explored, and the mechanism of NET formation was also studied by a series of in vitro and in vivo assays. Higher levels of NETs in plasma and tumor tissues were associated with dismal outcome in patients with DLBCL. Furthermore, we identified NETs increased cell proliferation and migration in vitro and tumor growth and lymph node dissemination in vivo. Mechanistically, DLBCL-derived IL8 interacted with its receptor (CXCR2) on neutrophils, resulting in the formation of NETs via Src, p38, and ERK signaling. Newly formed NETs directly upregulated the Toll-like receptor 9 (TLR9) pathways in DLBCL and subsequently activated NFκB, STAT3, and p38 pathways to promote tumor progression. More importantly, disruption of NETs, blocking IL8-CXCR2 axis or inhibiting TLR9 could retard tumor progression in preclinical models. Our data reveal a tumor-NETs aggressive interaction in DLBCL and indicate that NETs is a useful prognostic biomarker and targeting this novel cross-talk represents a new therapeutic opportunity in this challenging disease.

Human bone marrow-derived mesenchymal stem cells promote the growth and drug-resistance of diffuse large B-cell lymphoma by secreting IL-6 and elevating IL-17A levels

BackgroundThe drug-resistance and relapse of diffuse large B-cell lymphoma (DLBCL), which are related to mesenchymal stem cells (MSCs), have become increasingly common. However, the underlying mechanisms remain elusive.MethodsCCK 8 assay, colony formation assay, and xenograft mouse model were used to investigate the effects of hBMSCs on DLBCL growth. Immunohistochemistry, qRT-PCR, and ELISA were used to study the expressions of IL-6 and IL-17A. Flow cytometry was used to analyze Th17 cells and Treg cells expressions. Western blot analysis, microarray analysis, and bioinformatics analysis were used to analyze the pathways of IL-6 or IL-17A mediated DLBCL growth.ResultsHBMSCs promoted DLBCL growth by secreting IL-6 in vitro and in vivo and simultaneously upregulating IL-17A in vitro. IL-6 and IL-17A synergistically promoted the growth and drug-resistance of DLBCL cells by protecting them from spontaneous or drug-induced apoptosis in vitro. IL-6 or IL-17A activated the JAK2/STAT3 pathway or upregulated cyclin D2 via activation of PI3K/Akt signaling in vitro, respectively.ConclusionsThe present results indicated that hBMSCs might have a “dual effect” on promoting DLBCL progression and drug-resistance by secreting IL-6 and upregulating IL-17A. IL-6, IL-17A, p-STAT3, p-Akt or cyclin D2 may be potential molecular targets for overcoming drug-resistance in patients with relapsed or refractory DLBCL.

CUL4B regulates autophagy via JNK signaling in diffuse large B-cell lymphoma

ABSTRACT Aberrant expression of CUL4B was identified in various types of solid cancers. Cumulative evidences support the oncogenic role of CUL4B in cancers, including regulation of cell proliferation and signal transduction. However, its clinical value and potential pathogenic mechanism in diffuse large B-cell lymphoma (DLBCL) have not been described previously. Therefore, we hypothesize that overexpressed CUL4B may contribute to the pathogenesis of DLBCL. The aim of this study is to assess the expression and the biological function of CUL4B in DLBCL progression. In our study, CUL4B overexpression was observed in DLBCL tissues, and its upregulation was closely associated with poor prognosis in patients. Furthermore, the functional roles of CUL4B was detected both in vitro and in vivo. We demonstrated that silencing CUL4B could not only induce cell proliferation inhibition, cell cycle arrest, and motility attenuation of DLBCL cells in vitro, but also decrease tumor growth in DLBCL xenografts mice. In addition, we identified that CUL4B may act as a potent inductor of JNK phosphorylation in regulation of autophagy. Our findings demonstrated a significant role of CUL4B in the development and progression of DLBCL. CUL4B may act as a useful biomarker and a novel therapeutic target in DLBCL.

Identification of B-cell translocation gene 1-controlled gene networks in diffuse large B-cell lymphoma: A study based on bioinformatics analysis.

B-cell translocation gene 1 (BTG1) is a member of the BTG/transducer of Erb family. The present study evaluated the impact of BTG1 gene expression on the clinical outcome of diffuse large B-cell lymphoma (DLBCL) and investigated potential mechanisms using the Gene Expression Omnibus (GEO) database. The gene expression profile datasets GSE31312, GSE10846, GSE65420 and GSE87371 were downloaded from the GEO database. BTG1 expression and clinicopathological data were obtained from the GSE31312 dataset. In 498 cases, the expression of BTG1 in DLBCL was associated with treatment response (χ2=19.020; P<0.001) and International Prognostic Index score (χ2=5.320; P=0.025). Using the Kaplan-Meier method, it was identified that the expression of BTG1 was associated with overall survival (OS) and progression-free survival (PFS) times. Univariate and multivariate Cox regression analysis demonstrated that BTG1 was an independent predictive factor for OS and PFS. From the overlapping analysis of 407 BTG1-associated genes and 22,187 DLBCL-associated genes, 401 genes were identified as BTG1-associated DLBCL genes. Pathway analysis revealed that BTG1-associated DLBCL genes were associated with cancer progression and DLBCL signaling pathways. Subsequently, a protein-protein interaction network was constructed of the BTG1-associated genes, which consisted of 235 genes and 601 interactions. Additionally, 24 genes with high degrees in the network were identified as hub genes, which included genes associated with ‘ribosome’ [ribosomal protein (RP) L11, RPL3, RPS29, RPL19, RPL15 and RPL12], ‘cell cycle’ (ubiquitin carboxyl extension protein 52, ATM and Ras homolog family member H), ‘mitogen-activated protein kinase pathway’ (mitogen-activated protein kinase 1), ‘histone modification’ (ASH1-like protein) and ‘transcription/translation’ (eukaryotic translation initiation factor 3 subunit E, eukaryotic translation elongation factor 1 δ, transcription termination factor 1, cAMP responsive element binding protein 1 and RNA polymerase II subunit F). In conclusion, BTG1 may serve as a predictive biomarker for DLBCL prognosis. Additionally, bioinformatics analysis indicated that BTG1 may exhibit key functions in the progression and development of DLBCL.

Gene expression profiles analysis identifies a novel two-gene signature to predict overall survival in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematologic malignancy, however, specific tumor-associated genes and signaling pathways are yet to be deciphered. Differentially expressed genes (DEGs) were computed based on gene expression profiles from GSE32018, GSE56315, and The Cancer Genome Atlas (TCGA) DLBC. Overlapping DEGs were then evaluated for gene ontology (GO), pathways enrichment, DNA methylation, protein–protein interaction (PPI) network analysis as well as survival analysis. Seventy-four up-regulated and 79 down-regulated DEGs were identified. From PPI network analysis, majority of the DEGs were involved in cell cycle, oocyte meiosis, and epithelial-to-mesenchymal transition (EMT) pathways. Six hub genes including CDC20, MELK, PBK, prostaglandin D2 synthase (PTGDS), PCNA, and CDK1 were selected using the Molecular Complex Detection (MCODE). CDC20 and PTGDS were able to predict overall survival (OS) in TCGA DLBC and in an additional independent cohort GSE31312. Furthermore, CDC20 DNA methylation negatively regulated CDC20 expression and was able to predict OS in DLBCL. A two-gene panel consisting of CDC20 and PTGDS had a better prognostic value compared with CDC20 or PTGDS alone in the TCGA cohort (P=0.026 and 0.039). Overall, the present study identified a set of novel genes and pathways that may play a significant role in the initiation and progression of DLBCL. In addition, CDC20 and PTGDS will provide useful guidance for therapeutic applications.

Diffuse Large B Cell Lymphoma in an Adolescent: A Challenging Case with Intracerebral and Retinal Involvement

Diffuse large B-cell lymphoma (DLBCL) is a rare lymphoma type in pediatric patients with usually good prognosis. We present the difficult diagnostic procedure and the complicated course in an adolescent patient with DLBCL.Case report: The 16-year-old female patient presented with a history of fever episodes during the past 3 weeks, hepatosplenomegaly, leukopenia, anemia, hypertriglyceridemia, massive hyperferritinemia and elevation of lactate dehydrogenase. Leukemia was excluded by bone marrow puncture. Initially, no significant lymph node enlargement was detected by imaging of the mediastinum and the abdomen but a pathological signal of the bone marrow could be observed in magnetic resonance imaging (MRI). Hemophagocytic lymphohistiocytosis (HLH) was diagnosed and the patient was treated with prednisone 0.5 mg/kg per day. The overall condition and the laboratory values improved but deteriorated on cessation of the steroid treatment. The result of the bone marrow biopsy then revealed an infiltration of a malignant B-cell lymphoma. Diagnosis of a DLBCL, Non-GCB-type with a BCL6-break was subsequently made in a biopsy of a lymphnode which had shown increased uptake in PET scan. Treatment according to the current NHL-BFM registry including administration of Rituximab was started. The course of disease was complicated by HLH symptoms with persistent fever and pancytopenia, renal failure, severe Methotrexate excretion failure requiring Carboxypeptidase treatment and Herpes virus encephalitis. After recovery progression of DLBCL was found and the treatment was switched to DA-EPOCH regimen and repeated systemic Rituximab administrations. Five months after diagnosis the girl suffered from visual impairment and convulsions. Ophthalmoscopy revealed retinal lesions and intracerebral lesions were detected by MRI but no abnormalities of cerebrospinal fluid (CSF) were found. Biopsy of a brain lesion confirmed cerebral involvement of DLBCL. Intensified treatment included high- dose steroids, cranio-spinal irradiation, high-dose Cytarabin and intrathecal Rituximab. Response of the cerebral lesions but no improvement of the visual impairment was found. Salvage treatment with Gemcitabin, Cisplatin and Rituximab was administered because of persistent nodal and extranodal lymphoma manifestations. The patient finally died due to refractory disease and severe bone marrow failure.Conclusion: This case of DLBCL in an adolescent girl is extraordinary in many ways. DLBCL, especially the non-GCB type, is a rare disorder in pediatric patients. Predominant bone-marrow involvement has been described in only few older patients with non-GCB-type DLBCL and mostly aggressive clinical course. HLH as a presenting feature is rather observed in T- or NK-cell lymphoma than in DLBCL. Finally, central nervous system (CNS) relapse occurs in only about 6% of adults with DLBCL regardless of CNS prophylaxis. Retinal and intraparenchymal CNS involvement without abnormalities in the CSF has been typically described for primary CNS lymphoma in adults. All these conditions have contributed to the adverse outcome in our patient. DisclosuresNo relevant conflicts of interest to declare.

Human Bone Marrow-Derived Mesenchymal Stem Cells Promote the Growth and Drug-Resistance of Diffuse Large B-Cell Lymphoma by Secreting IL-6 and Elevating IL-17A Levels

Background: The drug-resistance and relapse of diffuse large B-cell lymphoma (DLBCL), which are related to mesenchymal stem cells (MSCs), have become increasingly common. However, the underlying mechanisms remain elusive. Methods: CCK 8 assay, colony formation assay, and xenograft mouse model were used to investigate the effects of hBMSCs on DLBCL growth.Immunohistochemistry, qRT-PCR, and ELISA were used to study the expressions of IL-6 and IL-17A. Flow cytometry was used to analyze Th17 cells and Treg cells expressions. Western blot analysis, microarray analysis, and bioinformatics analysis were used to analyze the pathways of IL-6 or IL-17A mediated DLBCL growth. Findings: HBMSCs promoted DLBCL growth by secreting IL-6 in vitro and in vivo and simultaneously upregulating IL-17A in vitro. IL-6 and IL-17A synergistically promoted the growth and drug-resistance of DLBCL cells by protecting them from spontaneous or drug-induced apoptosis in vitro. IL-6 or IL-17A activated the JAK2/STAT3 pathway or upregulated cyclin D2 via activation of PI3K/Akt signaling in vitro, respectively. Interpretation: The present results indicated that hBMSCs might have a dual effect on promoting DLBCL progression and drug-resistance by secreting IL-6 and upregulating IL-17A. IL-6, IL-17A, p-STAT3, p-Akt or cyclin D2 may be potential molecular targets for overcoming drug-resistance in patients with relapsed or refractory DLBCL. Funding Statement: This work was supported by the Natural Science Foundation of Guangdong Province, China (2018A0303130167); the Guangzhou Planned Project of Science and Technology, China (201707010279, 201300000100, and 201704020105); the Guangdong Medical Science and Research Foundation, China (A2018258); the Guangzhou General Science and Technology Project of Health and Family Planning, China (20181A011011). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This research was approved by the Ethics Committee of Guangzhou First People’s Hospital (K-2017-066-02). Written informed consent was obtained from all participants or their families. All animal experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at Guangzhou First People's Hospital, Guangzhou Medical University.

STAT3-coordinated migration facilitates the dissemination of diffuse large B-cell lymphomas

The motile characteristics and mechanisms that drive the dissemination of diffuse large B-cell lymphoma (DLBCL) are elusive. Here, we show that DLBCL initiates dissemination through activating STAT3-mediated amoeboid migration. Mechanistically, STAT3 activates RHOH transcription, which competes with the RhoGDP dissociation inhibitor RhoGDIγ to activate RhoA. In addition, activated STAT3 regulates microtubule dynamics and releases ARHGEF2 to activate RhoA. Both the JAK inhibitor ruxolitinib and the microtubule stabilizer Taxol suppress DLBCL cell dissemination in vivo. A clinical DLBCL sample analysis shows that STAT3-driven amoeboid movement is particularly important for the transition from stage I to stage II. This study elucidates the mechanism of DLBCL dissemination and progression and highlights the potential of combating advanced DLBCL with a JAK/STAT inhibitor or microtubule stabilizer to reduce DLBCL motility; these findings may have a great impact on the development of patient-tailored treatments for DLBCL.

Bortezomib inhibited the progression of diffuse large B-cell lymphoma via targeting miR-198.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, which is an aggressive malignancy with high variance of clinical features and response to the treatment. The proteasome inhibitor bortezomib (BTZ) has been demonstrated to suppress the progression of DLBCL, however, the underlying molecular mechanisms by which BTZ regulates the growth of DLBCL cells remain largely unknown. Increasing evidence has suggested that microRNAs (miRNAs) are novel targets of anti-cancer drugs to modulate the progression of cancers. Here, we showed BTZ treatment significantly inhibited the proliferation of DLBCL CRL-2630 cells. Mechanistically, exposure of BTZ up-regulated the expression of miR-198 in DLBCL cells. Depletion of miR-198 significantly reversed the inhibitory effect of BTZ on the proliferation of CRL-2630 cells. To further characterize the involvement of miR-198 in BTZ-induced growth defects of CRL-2630 cells, the downstream targets of miR-198 were predicted with the bioinformatics tools. The results showed that miR-198 bound the 3'-untranslated region (UTR) of the high mobility group AT-hook 1 (HMGA1) and suppressed the expression of HMGA1 in DLBCL cells. Consistently, BTZ treatment decreased the level of HMAG1 and inhibited the migration of DLBCL cells. Our results provided the possible mechanism by which BTZ suppressed the growth of DLBCL cells.

A Preliminary Study on Metadherin as a Potential Marker for Progression of Diffuse Large B Cell Lymphoma.

To determine if Metadherin (MTDH) expression levels are positively correlated with the clinical stage of diffuse large B-cell lymphoma (DLBCL) based on MTDH being highly expressed in other type of tumors including melanoma, malignant glioma, breast cancer, and hepatocellular carcinoma. In this study, we investigated the pathologic significance of MTDH and its potential in predicting DLBCL outcomes. Tissue samples from 50 patients with DLBCL and 22 patients with lymph node reactive hyperplasia were collected and evaluated using immunohistochemical staining, microscopy, and western blotting. The Kaplan-Meier method and Cox regression model were used for survival analysis of patients. Our results show that the overexpression of the MTDH protein in tissues was observed in 66% of patients with DLBCL, whereas it was not overexpressed in the patients with reactive hyperplastic lymph nodes. While there was no correlation between MTDH overexpression with age, sex, presence of B symptoms, and lactate dehydrogenase (LDH) levels in patients with DLBCL, this parameter was positively correlated with clinical stages. Moreover, MTDH-negative patients had significantly better prognoses compared with the MTDH-positive patients. Our preliminary study indicates that MTDH may play an important role in the development of DLBCL, and that MTDH overexpression is potentially associated with the clinical progression of DLBCL. In addition, high expression levels of MTDH in tissues was correlated with a poorer prognosis for patients with DLBCL. As such, MTDH may be a potential therapeutic target for specific therapy. However, research on a larger group of patients is needed to verify these preliminary results.

Abstract 1886: A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy

Abstract The purpose of this project is to investigate the feasibility of using small molecules targeting cap-dependent translation to sensitize B cell lymphoma cells to killing by BCL-2 inhibitors (venetoclax/ ABT-199). We hypothesized that diffuse large B-cell lymphoma (DLBCL) cell survival requires cap-dependent translation, which is facilitated by eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interaction and is promoted by mTOR complex 1 (mTORC1). We used the compound SBI-756, a novel inhibitor targeting the scaffolding protein eIF4G (1). SBI-756 is more potent and selective than previously reported compounds targeting the eIF4E cap-binding protein, or compounds targeting eIF4A helicase. By treating DLBCL cells (germinal center B-cell subtype) with SBI-756 (sub μM concentrations), we discovered profound synergistic induction of apoptosis when combined with venetoclax. Cell viability was reduced even more compared to mTOR kinase inhibitor (TOR-KI) treatment combination. Moreover, SBI-756 was found to reduce the viability (IC50 = 653nM) of VAL lymphoma cells lacking 4E-BP1, while VAL were insensitive to TOR-KI treatment. By using Proximity Ligation Assay (PLA) we showed that SBI-756 treatment prevents eIF4E-eIF4G association in intact lymphoma cells. Dual luciferase assays validated this and indicated a dose-dependent reduction in cap-dependent translation following SBI-756 treatment. Also, Western blot analyses confirmed that SBI-756 treatment did not change mTOR substrates' phosphorylation, indicating that the SBI-756 effect is specific to preventing the eIF4E-eIF4G interaction. Furthermore, SBI-756 treatment reduced polysome formation with a corresponding increase in abundance of monosomes. Lastly, induced expression of 4E-BP1 mutant, which irreversibly binds eIF4E, sensitized DLBCL cells to venetoclax treatment to a similar extent as TOR-KI treatment. Ongoing studies will investigate: (1) The molecular mechanism by which translation inhibition by SBI-756 synergizes with venetoclax to prime DLBCL cells toward apoptosis, and (2) pharmacokinetic and pharmacodynamic effects of venetoclax and SBI-756 treatment on DLBCL progression in vivo. Hence, this project highlights a novel combination for treatment of aggressive lymphomas, and establishes its efficacy using preclinical models. Reference: 1. Feng Y., et al. SBI-0640756 attenuates the growth of clinically unresponsive melanomas by disrupting the eIF4F translation initiation complex. Cancer Res 2015;75(24):OF1-8. Citation Format: Lee-or Herzog, Nancy Nguyen, Honyin Chiu, Sharmila Mallya, Ze'ev A. Ronai, David A. Fruman. A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1886.

Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway.

Diffuse large B-cell lymphoma (DLBCL) is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel prognostic markers and therapeutic targets of DLBCL. Recent studies have shown that long non-coding RNAs (lncRNAs) play an important role in the development of cancer. By using the next generation HiSeq sequencing assay, we determined lncRNAs exhibiting differential expression between DLBCL patients and healthy controls. Then, RT-qPCR was performed for identification in clinical samples and cell materials, and lncRNA PANDA was verified to be down-regulated in DLBCL patients and have considerable diagnostic potential. In addition, decreased serum PANDA level was correlated to poorer clinical outcome and lower overall survival in DLBCL patients. Subsequently, we determined the experimental role of lncRNA PANDA in DLBCL progression. Luciferase reporter assay and chromatin immunoprecipitation assay suggested that lncRNA PANDA was induced by p53 and p53 interacts with the promoter region of PANDA. Cell functional assay further indicated that PANDA functioned as a tumor suppressor gene through the suppression of cell growth by a G0/G1 cell cycle arrest in DLBCL. More importantly, Cignal Signal Transduction Reporter Array and western blot assay showed that lncRNA PANDA inactivated the MAPK/ERK signaling pathway. In conclusion, our integrated approach demonstrates that PANDA in DLBCL confers a tumor suppressive function through inhibiting cell proliferation and silencing MAPK/ERK signaling pathway. Thus, PANDA may be a promising therapeutic target for patients with DLBCL.

MYC is a positive regulator of choline metabolism and impedes mitophagy-dependent necroptosis in diffuse large B-cell lymphoma

The activation of oncogenes can reprogram tumor cell metabolism. Here, in diffuse large B-cell lymphoma (DLBCL), serum metabolomic analysis revealed that oncogenic MYC could induce aberrant choline metabolism by transcriptionally activating the key enzyme phosphate cytidylyltransferase 1 choline-α (PCYT1A). In B-lymphoma cells, as a consequence of PCYT1A upregulation, MYC impeded lymphoma cells undergo a mitophagy-dependent necroptosis. In DLBCL patients, overexpression of PCYT1A was in parallel with an increase in tumor MYC, as well as a decrease in serum choline metabolite phosphatidylcholine levels and an International Prognostic Index, indicating intermediate–high or high risk. Both in vitro and in vivo, lipid-lowering alkaloid berberine (BBR) exhibited an anti-lymphoma activity through inhibiting MYC-driven downstream PCYT1A expression and inducing mitophagy-dependent necroptosis. Collectively, PCYT1A was upregulated by MYC, which resulted in the induction of aberrant choline metabolism and the inhibition of B-lymphoma cell necroptosis. Referred as a biomarker for DLBCL progression, PCYT1A can be targeted by BBR, providing a potential lipid-modifying strategy in treating MYC-High lymphoma.

MicroRNA-26a/cyclin-dependent kinase 5 axis controls proliferation, apoptosis and in vivo tumor growth of diffuse large B-cell lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma. Despite a favorable therapeutic response to first-line chemo-immunotherapy, still 30–40% of patients is refractory, or relapse after this treatment. Thus, alternative strategies must be sought. Previous studies have indicated that cyclin-dependent kinase 5 (CDK5), a serine/threonine protein kinase, is involved in tumor development and progression, and it may represent a potential therapeutic target. However, its role in modulating DLBCL growth and progression remains largely unexplored. In this study, we show that CDK5 and its activator, cyclin-dependent kinase 5 activator 1 (CDK5R1 or p35), are overexpressed in DLBCL cell lines and that signal transducer and activator of transcription 3 (STAT3) phosphorylation and activity is dependent on CDK5 expression in DLBCL. Using public data sets, we also demonstrate that patients with DLBCL show a higher expression of CDK5 compared with healthy individuals. By using loss-of-function approaches, we demonstrate that CDK5’s activity regulates proliferation and survival of DLBCL cells. MicroRNAs (miRNAs or miRs) are small noncoding RNAs that negatively regulating gene expression and are involved in cancer initiation and progression. We identify miR-26a as direct regulator of p35 expression and CDK5 activity. We show that miR-26a expression is lower in DLBCL cell lines compared to B lymphocytes and that its ectopic expression leads to a drastic reduction of DLBCL tumor growth in vivo and decreased proliferation, cell-cycle progression, and survival in vitro. Remarkably, concomitant overexpression of a 3′-UTR-truncated form of p35 promoted tumor growth in vivo and cell proliferation, cell-cycle progression, and cell survival in vitro. In conclusion, these results demonstrate an important role for miR-26a and CDK5 together in the survival and growth of DLBCL cells, suggesting the existence of potential novel therapeutic targets for the treatment of DLBCL.