scholarly journals A Single-Cell Atlas of Nonhematopoietic Cells in Human Lymph Node and Lymphoma Reveals Landscape of Stromal Remodeling

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 447-447
Author(s):  
Yoshiaki Abe ◽  
Mamiko Sakata-Yanagimoto ◽  
Manabu Fujisawa ◽  
Hiroaki Miyoshi ◽  
Yasuhito Suehara ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lymph Node ◽  
B Cell ◽  
Single Cell ◽  
Stromal Cells ◽  
Clustering Analysis ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Human Lymph Node ◽  
Reticular Cells

Abstract Background: Activities of nonhematopoietic cells (NHCs) reportedly underlie lymphomagenesis. In follicular lymphoma (FL), mesenchymal stromal cells (SCs) including follicular dendritic cells (FDCs) have been shown to facilitate FL expansion. However, comprehensive understanding of lymphoma NHC activities have been hampered by indefinite NHC heterogeneity even in normal human lymph node (LN). Indeed, human LN blood endothelial cells (BECs) and non-endothelial stromal cells (NESCs) have not been analyzed at single-cell resolution. Here, we aimed to construct a single-cell atlas of NHCs in human LN applicable to lymphoma researches. We also sought to reveal the landscape of stromal remodeling in lymphomas, particularly in FL, to advance understanding of stromal contributions in lymphomagenesis. Methods: We prospectively performed single-cell RNA sequencing of NHCs (>100,000 cells) extracted from 27 human samples including metastasis-free LN (MFLN; n=9), nodal FL (n=10), peripheral T-cell lymphoma (PTCL; n=5), and diffuse large B-cell lymphoma transformed from FL (tDLBCL; n=3). Data from MFLN samples were used for the construction of NHC atlas. Immunofluorescence (IF) staining was performed to investigate the existence and topological localizations of each NHC subcluster in the LN. Using the NHC atlas, we performed comprehensive comparative analysis with FL NHCs by differentially-expressed gene (DEG) and intercellular ligand-receptor analyses. We also investigated the prognostic impact of putative stroma-derived biomarkers using deposited microarray data of FL patients. Finally, we examined the applicability of the atlas to NHCs from other lymphoma subtypes by analyzing PTCL and tDLBCL NHCs. Data analysis was performed through multiple pipelines including Seurat, Monocle3, and CellphoneDB. Results: Graph-based clustering analysis revealed that the transcriptional features of NHC subpopulations in MFLN are detectable in FL NHCs. Unsupervised sub-clustering analysis of BECs, lymphatic endothelial cells (LECs), and NESCs revealed 10, 8, and 12 subclusters, respectively, including some lacking mouse counterpart. IF staining successfully identified each NHC subcluster and its localization in the LN. In FL NHCs, the proportion of arterial BEC subclusters markedly increased relative to MFLN, while the proportion of LECs decreased. In FL NESCs, the proportion of marginal reticular cells (MRCs) as well as FDCs greatly increased. DEG analysis revealed that the greatest changes in gene expression occurs in NESC subclusters, particularly in MRCs, T-zone reticular cells (TRCs), pericytes, and FDCs. Notably, in some NESC subclusters, we observed marked upregulation of genes relevant to solid cancers but previously not described in lymphomas (e.g. POSTN, EGFL6, and FAP). Combined interactome and DEG analysis revealed 60 FL-specific interactions between NHC subclusters and malignant B cells. For example, interactions mediated through stroma-derived CD70 were enhanced at medullary SC subclusters and SCs at LN capsule adventitia. Additionally, the CCR7-CCL19 interaction and interactions via B-cell activating factor (BAFF) were unexpectedly upregulated at non-TRC SC and medullary SC subclusters, respectively. Also, the CXCL13-CXCR5 axis was highly activated in MRCs, collectively indicating that non-FDC SCs vigorously participate in FL cell expansion and/or infiltration into extra-follicular lesions. Some intercellular interactions were functionally validated by in vitro binding assays. Based on this dataset, we identified putative stroma-derived biomarkers linked to unfavorable prognosis in FL patients including TDO2, encoding immune-modulators, and LY6H and LOX, tip cell markers. We finally confirmed that NHC subclusters identified in our atlas were also detectable in NHCs of more aggressive lymphoma subtypes including PTCL and tDLBCL. Notably, we found that extra-follicular SCs had further differentiated into follicular SCs in tDLBCL, likely representing a terminal form of stromal remodeling in FL. Conclusion: We constructed a comprehensive single-cell atlas of NHCs in human LN highly applicable to lymphoma NHC researches and revealed a total of 30 NHC subclusters. Our study largely updates NHC taxonomy in LNs and provides a rich resource and deeper insights into lymphoma biology, a contribution that should advance lymphoma management and therapy. Figure 1 Figure 1. Disclosures Usuki: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Research Funding; Celgene K.K.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Nippon-Boehringer-Ingelheim Co., Ltd.: Research Funding; Mundipharma K.K.: Research Funding; Amgen-Astellas Biopharma K.K.: Research Funding; Nippon-Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Kyowa-Kirin Co., Ltd.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Research Funding, Speakers Bureau; Yakult Honsha Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Research Funding; Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Apellis Pharmaceuticals, Inc.: Research Funding; AbbVie GK: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Incyte Biosciences Japan G.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sanofi K.K.: Speakers Bureau; Amgen K.K.: Research Funding.

scholarly journals Unravelling the Heterogeneity of Mantle Cell Lymphoma Ecosystem By Single Cell RNA Sequencing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4118-4118
Author(s):  
Makhdum Ahmed ◽  
Hui Guo ◽  
Shaojun Zhang ◽  
Lalit Sehgal ◽  
Preetesh Jain ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Single Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Clonal Evolution ◽  
Advisory Committees ◽  
Cell Clones ◽  
Mantle Cell

Abstract Background: Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma that is incurable. MCL has a complex ecosystem of malignant B-cells and stromal and immune cells that play a supporting role for tumor growth, ultimately leading to the potential re-emergence of the disease. The tumor microenvironment has been reported as a crucial factor in MCL pathogenesis and progression. Thus, if we can identify the tumor microenvironment components and define the characteristics of malignant and non-malignant cells, this will pave the way for studying clonal evolution of MCL in vivo. Methods: Both pre- and post-treatment, fresh tumor biopsy samples of MCL were obtained. The cells were dissociated and re-suspended in PBS with >10% serum. A final concentration of 1,200 cells/uL were used for single cell sorting in the chromium system (10X Genomics, California). We sequenced the mRNA in the NextSeq 500 platform. All analysis was conducted using R-programming language (version 3.4). Results: From four MCL patients (L1-L4), we obtained 9,400 cells. Three of the four samples were collected through apheresis (i.e., L1-L3), and one sample (i.e., L4) from surgical biopsy of the involved lymph node. One patient had known TP53 mutated status (i.e., L1) and another patient had CCND1 translocation (i.e., L2). From the apheresis samples (L1-L3), the proportion of lymphocytes was 87%, 68% and 65%. We identified 10 defined clusters of cells based upon their gene expression from all four samples. Six of the 10 clusters were clonal B-cells with strong expression of CCND1, CD79A and CD79B. We also identified clonal T-cells (both CD8+ and CD4+) and monocyte/macrophage clusters. SOX11 expression was absent in one B-cell clone, indicating this clone may be SOX11-negative MCL. The monocyte-macrophage cluster demonstrated strong BCL2 expression, which was not expressed by the B-cells clones. CD19 expression was ubiquitous among the B-cell clones but weaker as compared with other B-cell markers. When the signaling was compared among the four samples, the chemo-resistant cells (sample L1) demonstrated upregulation of NOTCH1 signaling, DNA-damage repair, interferon-alpha response, MYC targets and the HIF1A pathway. Two cell clones did not express any canonical markers and were not identified as a defined cluster. Conclusions: We identified meaningful sub-populations of MCL that define the tumor microenvironment. There is considerable inter-tumor and intra-tumor heterogeneity of MCL at a single cell resolution, which indicates that developing a uniform treatment regimen may prove to be difficult. Ubiquitous expression of CD79A or CD79B may help guiding precision medicine such as the development of novel CAR-T cell therapy. Longitudinal follow up of the same patients may define clonal evolution of MCL and unravel the spatio-temporal interplay. Figure. Figure. Disclosures Wang: AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Novartis: Research Funding; Acerta Pharma: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding.

scholarly journals Mass Cytometry Based Classification of Inter- and Intra-Tumoral Heterogeneity in Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3908-3908
Author(s):  
Manabu Kusakabe ◽  
Xuehai Wang ◽  
Guillermo Simkin ◽  
Justin Meskas ◽  
Chaoran Zhang ◽  
...  
Keyword(s):  
Lymph Node ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: Recent work in both hematologic malignancies and solid tumors has supported the notion that human cancers exhibit marked intra-tumoral heterogeneity (ITH). Results from next generation sequencing (NGS) studies support that subclonal DNA mutations underlie genotypic ITH within a single tumor since the majority of sequence variants are present in only 5-50% of reads for a given tumor sample, and single cell analyses have shown that individual tumor subclones may be ancestrally related in a complex branching hierarchy, suggesting that therapy failures and progressive disease likely arise by Darwinian selection for more aggressive or therapy resistant clones. It has become increasingly clear that it will be important to understand the multi-clonal structure of tumors in order to treat them more effectively. In this study, we sought to use time-of-flight mass cytometry (CyTOF) to explore clonal phenotypic substructure in diffuse large B-cell lymphoma (DLBCL), a diagnostic entity notorious for clinical heterogeneity. Methods: We examined viably frozen single cell suspensions from diagnostic lymph node biopsy samples received for flow cytometric analysis at the BC Cancer Agency. We have thus far acquired CyTOF data from 25 cases of DLBCL using a two-tube, 40-parameter panel encompassing a total of 58 different markers including both surface and intracellular antigens that were selected to reveal heterogeneity within the malignant B-cell population. For each sample acquisition, we included "spiked-in" control cells from pooled reactive (non-malignant) lymph node samples to control for staining variation between antibody/reagent lots and also run-to-run CyTOF instrument drift, facilitated by a CD45 antibody "barcoding" approach. We analyzed the data using a combination of viSNE, Isomap, and PhenoGraph analysis packages. Results: Analysis of individual tumor samples readily distinguished between malignant and residual normal B-cell populations, and also revealed distinct subpopulations among malignant cells of varying degrees of relatedness to one another. These subpopulations were then sorted from one another by conventional FACS from parallel vials of cryopreserved cells using lower dimensional sorting strategies derived from the 40-parameter CyTOF data. Sorted subpopulations will be analyzed by targeted amplicon sequencing for single nucleotide variants identified from whole exome sequencing data obtained from unsorted material to explore the hypothesis that these may represent genotypic subclones. 
Analysis of multiple tumor samples at once yielded several observations. First, B-cells from reactive lymph nodes and non-malignant B-cells within patient lymphoma specimens reproducibly cluster atop one another, indicating highly similar if not identical phenotypic profiles. Second, the majority of patient DLBCL tumors form cohesive individual clusters, separate and distinct from one another, suggesting that the 40-dimensional panel defines cell populations with sufficient resolution such that each patient's tumor can be uniquely identified. Third, individual DLBCL tumors do not aggregate in tight proximity with one another to the extent that we observe among patient follicular lymphoma (FL) samples, suggesting DLBCL represents a broader diversity of phenotypic classes. Fourth, there is local, but loose aggregation of ABC versus GCB subtypes, but there are also clear outliers and areas of intermingling between ABC and GCB tumors, as defined by immunohistochemistry. Finally, a subset of DLBCL tumors exhibit minor subpopulations that map apart from their corresponding "parent" tumor populations, but yet overlap one another, raising the possibility of divergent evolution away from (or alternatively convergent evolution towards) a common tumor archetype. Conclusions: 
Taken together, these observations support that novel information can be derived from CyTOF data with important implications for our understanding of both intra- and inter-tumoral heterogeneity in DLBCL. Disclosures Scott: Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

scholarly journals CG'806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broader and Greater Potency Than Ibrutinib Against Primary and Cultured Malignant B Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3503-3503
Author(s):  
Hongying Zhang ◽  
Andrea Local ◽  
Khalid Benbatoul ◽  
Peter Folger ◽  
Susan Sheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Malignancies ◽  
Apoptotic Effect ◽  
Btk Inhibitor ◽  
Induced Apoptosis

Abstract Mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) account for >70% of cases of B cell lymphoma. Targeting Bruton tyrosine kinase (BTK) with ibrutinib in B cell malignancies led to a paradigm shift in therapy. However, primary resistance to ibrutinib has been observed in about 30% MCL patients; more than 50% patients with CLL and MCL treated with ibrutinib discontinue treatment due to intolerance or emergence of resistant disease (Woyach et al., 2017; Shpilberg et al., 2018). CG'806 is an oral small molecule non-covalent pan-FLT3/pan-BTK inhibitor designed to address the shortcomings of ibrutinib. It is in development for acute myeloid leukemia (AML) and B cell lymphoma. CG'806 inhibited cell proliferation and induced apoptosis with a potency that was 50-6,000 times greater than that of ibrutinib when tested against 14 established malignant B-cell lines in vitro. When tested against 124 samples freshly isolated from the marrow of CLL patients the median IC50 for CG'806 was 0.11 µM and the median for ibrutinib was 4.09 µM, respectively, p<0.001). Since stromal-mediated signaling plays important roles in malignant B cell survival and chemoresistance, the apoptotic effect of CG'806 was further analyzed on cultured and primary malignant B cells in the presence of stromal cells. CG'806 produced similar dose-dependent apoptotic effect on Mino cells, an MCL cell line, in the absence or presence of human stromal HS5 cells indicating that its potency was not impaired by factors released by these stroma cells. Most importantly, CG'806 dose-dependently induced apoptosis in ibrutinib-refractory primary MCL samples in the presence of CD40L-expressing stromal cells (N=4). Whereas 0.1 µM and 1 µM CG'806 caused about 25% and 45% apoptotic cell death, respectively, 1 µM ibrutinib induced less than 10% cell death under the same culture conditions. CG'806 inhibited malignant B cell colony formation and migration towards SDF1α about 2-fold more effectively than ibrutinib. Given the role of activated B cell receptor (BCR) and NFκB pathways in lymphoma, CG'806 was tested for its ability to impair signaling in these pathways. CG'806 produced cell line dependent and dose/time dependent decreases in the phosphorylation of BTK, PLCγ2, PI3K, AKT, mTOR, PKC, and ERK, and reduced. These effects were correlated with induction of PARP cleavage and cell cycle arrest. We conclude that CG'806 inhibits driver and rescue pathways to directly and potently kill a broad range of malignant B cells, including both establish cell lines and freshly isolated patient samples, thereby distinguishing CG'806 from ibrutinib and supporting clinical development of CG'806 in patients with CLL and other B-cell malignancies intolerant, resistant, or refractory to ibrutinib. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Local:Aptose Biosciences, Inc: Employment. Benbatoul:Aptose Biosciences, Inc: Employment. Folger:Aptose Biosciences, Inc: Employment. Sheng:Aptose Biosciences, Inc: Employment. McLaughlin:Aptose Biosciences, Inc: Other: internship. Danilov:Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding. Tyner:Constellation: Research Funding; Aptose: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding. Howell:Aptose Biosciences, Inc: Research Funding. Rice:Aptose Biosciences, Inc: Equity Ownership.

scholarly journals FoxM1-Mediated Signaling Promotes the Progression of Mantle Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4176-4176
Author(s):  
Hui Guo ◽  
Yixin Yao ◽  
Hui Zhang ◽  
Elizabeth Lorence ◽  
Makhdum Ahmed ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Research Funding ◽  
Tumor Volume ◽  
Cell Lymphoma ◽  
Advisory Committees ◽  
Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients (Wang et al., NEJM, 2013). However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FoxM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FoxM1 has recently been classified as a human proto-oncogene and we have previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, we investigated the prognostic significance of FoxM1 and its potential as a new MCL therapeutic target. Methods We assessed the anti-MCL effects of targeting FoxM1 in both ibrutinib-sensitive and -resistant MCL cell lines and clinical specimens. Cell viability assays were conducted targeting FoxM1 with thiostrepton, a published FoxM1 inhibitor. The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FoxM1 inhibitor thiostrepton at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. Furthermore, we investigated the importance of FoxM1 signaling in tumor migration and adhesion in Jeko-1 WT and BTK KD (generated from Jeko-1 using CRISPR/Cas9) cells. Jeko-1 WT and two BTK KD variants were transiently transfected with control siRNA and siRNA against FoxM1 (siFoxM1) for 24 and 48 hours. Afterwards, cells were loaded into transwell migration inserts in the presence or absence of human stromal cells. Migration was evaluated by counting migrated cells and normalizing the migrated cells to migration in the absence of stromal cells. The in vivo efficacy of thiostrepton was evaluated in two cell line Jeko-1 and Jeko-1 BTK KD mouse xenograft models. Upon engraftment, thiostrepton was administered intravenously five consecutive days a week at 50 mg/kg, and tumor burden was assessed via the measurement of circulating human β2M levels and tumor volume. FoxM1 plasma levels in the xenografted mice were also evaluated using ELISA at 0, 10, 20, and 30 days. Results Inhibition of FoxM1 with thiostrepton reduced the cell viability of both ibrutinib-sensitive (Jeko-1; SP-49; PT-1; PT-2) cell lines and patient samples as well as ibrutinib-resistant MCL cells (Maver-1; Z138; Jeko-BTK KD 1 and 2; PTs 3-6) at half-maximal inhibitory concentration (IC50) in the micromolar range (IC50 = 1-3 μM) for the majority of tested cells and patient samples. siFoxM1 significantly reduced (P < 0.05) stromal cell-mediated migration of both Jeko-1 WT and BTK KD cells compared with the cells transfected with control siRNA. Moreover, siFoxM1-treated cells showed reduced levels of the migration- and adhesion-related proteins snail, vimentin, and N-cadherin compared with the control cells. Additionally, in comparison to vehicle-treated control mice, thiostrepton treatment significantly reduced tumor volume (36% and 17%, respectively) and β2M levels (71% and 79%, respectively) in Jeko-1 and Jeko-1 BTK KO xenografted mice at Day 30 of treatment regardless of BTK status. Lastly, thiostrepton treatment significantly suppressed the plasma levels of FoxM1 in both Jeko-1 (14%) and Jeko-1 BTK KO (11%) xenografted mice at Day 30 of treatment. Conclusion We have shown that FoxM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vivo studies. Therefore, exploring the role of FoxM1 in MCL disease progression and therapeutic resistance may lead to novel therapeutic breakthroughs to improve patient clinical outcomes. Disclosures Wang: MoreHealth: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria.

Single-Cell RNA Sequencing Identifies a Pseudo-Immune Differentiation Axis As the Main Source of Functional Heterogeneity in Follicular Lymphoma B-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 548-548
Author(s):  
Noudjoud Attaf ◽  
Inaki Cervera-Marzal ◽  
Laurine Gil ◽  
Chuang Dong ◽  
Jean-Marc Navarro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lymph Node ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
Research Funding ◽  
Affinity Maturation ◽  
Patient Specific ◽  
Specific Gene

Introduction Follicular Lymphoma (FL), the second most frequent lymphoma in adults, often presents as a disseminated disease at diagnosis. Despite a generally slow progression and a median overall survival of more than 15 years with current chemo-immunotherapies, FL patients often suffer from multiple relapses. Yet, the biological mechanisms promoting FL dissemination, progression and relapse are still poorly understood. FL, like most B-cell lymphomas, originates from germinal centers (GC) where B-cells physiologically undergo clonal expansion, antibody affinity maturation, and differentiation into antibody-producing plasma cells (PC) or recirculating memory (Mem) B-cells. Recently, we provided evidence that FL B-cells are not blocked in a GC B-cell state but might adopt new dynamic modes of functional diversity (Milpied et al., Nature Immunology 2018), yet the main sources of intratumoral heterogeneity within FL remained to be identified. Methods Frozen live cell suspensions were obtained from the CeVi collection of the Institute Carnot/Calym (ANR, France). We initially applied a plate-based 5'-end single-cell RNAseq (scRNAseq) method for deep integrative single-cell analyses of transcriptome, B-cell receptor (BCR) sequence, and surface phenotype on FACS-sorted FL B-cells (4 patients, lymph node biopsies) and their non-malignant counterparts (6 adult healthy donors, spleen and tonsil samples). We confirmed our findings on additional FL samples with high-throughput droplet-based 3'-end scRNAseq (9 patients, lymph node biopsies), and 5'-end scRNAseq paired with BCRseq (5 patients, lymph node biopsies). Custom and existing bioinformatics analysis pipelines were combined for quality control and cell filtering, dimensionality reduction (PCA, t-SNE, UMAP), clustering, pseudo-time analysis, BCR sequence analysis and integrative data analysis. We further validated our transcriptomic data with FACS-based surface and intracellular protein analysis (8 patients, lymph node biopsies). Results Consistent with our previous findings, FL B-cells were transcriptionally diverse, with most cells exhibiting a patient-specific gene expression profile distinct from PC, GC and Mem cells. Challenging the mainstream view of a differentiation blockade in FL, we identified rare FL B-cells carrying a PC-like profile (including low expression of MS4A1/CD20, high expression of XBP1, MZB1, PRDM1). PC-like FL B-cells expressed high levels of the tumor clonal BCR heavy and light chain mRNA, and BCR sequence phylogenetic analysis revealed that those cells did not branch out from a specific tumor subclone. Most importantly, we found that the molecular profiles of the vast majority of FL B-cells spanned a continuum of transitional states between proliferating GC-like and quiescent Mem-like gene expression states. Principal component analysis and pseudo-time reconstruction revealed that pseudo-immune differentiation axis was consistently the main source of intra-sample transcriptional heterogeneity. On top of cell cycle related genes, GC-like FL B-cells notably expressed AICDA, BCL6, RGS13, NANS, CD81, and CD38 genes. By contrast, Mem-like FL B-cells expressed CD44, GPR183, CD69, CXCR4, CCR7, SELL, KLF2, suggesting that those cells may not be confined to the FL follicles. Flow cytometry analysis of dissociated FL tumors confirmed that only the CD38hiCD81hi subset of FL B-cells (GC-like cells), expressed Ki67 and high levels of Bcl6, whereas only CD38negCD81neg FL B-cells (Mem-like cells) consistently contained CD44+ and GPR183+ cells. Conclusions Our study suggests that FL B-cells hijack the physiological GC differentiation process to dynamically alternate between GC-like and Mem-like states that might be responsible for FL progression and dissemination, respectively. We anticipate that such FL-specific clonal dynamics may be orchestrated by extrinsic signals delivered by tumor-infiltrating T cells. Disclosures Milpied: Innate Pharma: Research Funding; Institut Roche: Research Funding.

Indolent Mantle Cell Lymphoma: A Distinct Subgroup Characterized by Leukemic Phase Disease without Lymphadenopathy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3937-3937 ◽  
Author(s):  
Dong Chen ◽  
David S. Viswanatha ◽  
Clive S. Zent ◽  
Tait D. Shanafelt ◽  
Timothy G. Call ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymph Node ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Advisory Committees ◽  
Mantle Cell ◽  
Leukemic Phase

Abstract Abstract 3937 Poster Board III-873 Introduction Mantle cell lymphoma (MCL), defined by the pathognomic CCND1/IGH translocation, commonly presents with lymphadenopathy and has an aggressive clinical course with poor survival. However, a minority of patients with MCL present with lymphocytosis without substantial adenopathy. The clinical and pathologic features of this group of patients are not currently well understood. Methods Patients with monoclonal B-cell lymphocytosis, positive CCND1/IGH translocation status by FISH analysis and absence of lymphadenopathy were identified in our institutional B-cell lymphoproliferative disorder database. A comprehensive review of clinical, laboratory, and histopathologic features was conducted. Results of ancillary immunophenotypic, molecular genetic, and cytogenetic studies were reviewed. Univariate survival analysis was performed using the method of Kaplan and Meier. Result We identified a total of 35 patients meeting the selection criteria (10 female and 25 male; median age 66 yrs, range 41-86). The median absolute lymphocyte count at presentation was 8.7 × 109/L (range 2.7-180). Eleven patients subsequently developed adenopathy (MCL-LN) while 24 patients had persistent lymphocytosis without adenopathy (MCL-PB). There were no significant differences in age, gender, absolute leukocyte count, percent bone marrow involvement by lymphoma, or immunophenotypic features of the neoplastic B-cells between these 2 subgroups at initial evaluation. All 11 MCL-LN (100%) and 14 of the 24 MCL-PB patients (58%) had splenomegaly at presentation. Morphologic and immunophenotypic features in peripheral blood (n=35), bone marrow (n=34), lymph node (n=7), spleen (n=7) and other involved tissues (n=4) were typical of MCL in 34 patients. One patient had an unusual disease recurrence resembling hairy cell leukemia, but was retained in the analysis given the persistence of the CCND1/IGH abnormality and molecular evidence of clonal identity with previous specimens. All patients with MCL-LN and 12/24 (50%) of patients with MCL-PB received therapy including R-CHOP, cladribine, or rituximab. The remaining 12 MCL-PB patients (50%) were managed expectantly. Patients with MCL-PB (median survival of 156 months from diagnosis) had a significantly better survival than patients with MCL-LN (13 months) regardless of splenic involvement (P=0.0002) (Figure 1). Conclusion Among patients with MCL presenting in leukemic phase without adenopathy, we have identified 2 subgroups with distinct clinical outcomes. Patients who do not develop subsequent lymph node involvement (MCL-PB) have a generally more favorable clinical outcome. These findings imply fundamental differences in the pathobiology and molecular oncogenesis between these subgroups, suggesting the presence of distinct MCL entities. Disclosures: Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Gnentch: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospire: Research Funding; Polyphenon Pharma: Research Funding; Sanofi-Aventis: Research Funding; Biogenc-Ides: Membership on an entity's Board of Directors or advisory committees; Genmab: Membership on an entity's Board of Directors or advisory committees. Witzig:Novartis: Research Funding.

scholarly journals Tumor staging in a Beagle dog with concomitant large B-cell lymphoma and T-cell acute lymphoblastic leukemia

2021 ◽  
pp. 104063872110110
Author(s):  
Alessandro Ferrari ◽  
Marzia Cozzi ◽  
Luca Aresu ◽  
Valeria Martini
Keyword(s):  
Bone Marrow ◽  
Lymph Node ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
Tumor Staging ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Cell Acute Lymphoblastic Leukemia

An 8-y-old spayed female Beagle dog was presented with peripheral lymphadenomegaly. Lymph node cytology and flow cytometry led to the diagnosis of large B-cell lymphoma (LBCL). We detected minimal percentages of LBCL cells in peripheral blood and bone marrow samples. However, a monomorphic population of neoplastic cells different from those found in the lymph node was found in the bone marrow. T-cell acute lymphoblastic leukemia was suspected based on flow cytometric immunophenotyping. PCR for antigen receptor rearrangement (PARR) revealed clonal rearrangement of both B-cell and T-cell receptors, and the presence of both neoplastic clones in the lymph node, peripheral blood, and bone marrow. The dog was treated with multi-agent chemotherapy but died 46 d following diagnosis. Tumor staging and patient classification are needed to accurately establish a prognosis and select the most appropriate therapeutic protocol.

scholarly journals Plastin-3 is a diagnostic and prognostic marker for pancreatic adenocarcinoma and distinguishes from diffuse large B-cell lymphoma

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Guan-Hua Wu ◽  
Bing Wang ◽  
Yong-Jun Chen
Keyword(s):  
Lymph Node ◽  
B Cell ◽  
Cell Lymphoma ◽  
Quantitative Polymerase Chain Reaction ◽  
B Cell Lymphoma ◽  
Actin Binding ◽  
Rank Test ◽  
Large B Cell Lymphoma ◽  
Tissue Specimens ◽  
Large B Cell

Abstract Background Altered Plastin-3 (PLS3; an actin-binding protein) expression was associated with human carcinogenesis, including pancreatic ductal adenocarcinoma (PDA). This study first assessed differentially expressed genes (DEGs) and then bioinformatically and experimentally confirmed PLS3 to be able to predict PDA prognosis and distinguish PDA from diffuse large B-cell lymphoma. Methods This study screened multiple online databases and revealed DEGs among PDA, normal pancreas, diffuse large B-cell lymphoma (DLBCL), and normal lymph node tissues and then focused on PLS3. These DEGs were analyzed for Gene Ontology (GO) terms, Kaplan–Meier curves, and the log-rank test to characterize their association with PDA prognosis. The receiver operating characteristic curve (ROC) was plotted, and Spearman’s tests were performed. Differential PLS3 expression in different tissue specimens (n = 30) was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results There were a great number of DEGs between PDA and lymph node, between PDA and DLBCL, and between PDA and normal pancreatic tissues. Five DEGs (NET1, KCNK1, MAL2, PLS1, and PLS3) were associated with poor overall survival of PDA patients, but only PLS3 was further verified by the R2 and ICGC datasets. The ROC analysis showed a high PLS3 AUC (area under the curve) value for PDA diagnosis, while PLS3 was able to distinguish PDA from DLBCL. The results of Spearman's analysis showed that PLS3 expression was associated with levels of KRT7, SPP1, and SPARC. Differential PLS3 expression in different tissue specimens was further validated by RT-qPCR. Conclusions Altered PLS3 expression was useful in diagnosis and prognosis of PDA as well as to distinguish PDA from DLBCL.

Sign in / Sign up

Export Citation Format

Share Document