Integrated Analysis Of Genomic and Gene Expression Profiles In Follicular Lymphoma Reveals Subsets and Driver Genes Of Potential Microenvironmental Importance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2487-2487
Author(s):  
Ondrej Havranek ◽  
Jason R. Westin ◽  
Min Zhang ◽  
Seema Rawal ◽  
Larry W. Kwak ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Immune Cells ◽  
Copy Number ◽  
Integrated Analysis ◽  
Driver Genes ◽  
Gene Modules ◽  
Cell Fractions

Abstract Background The immune microenvironment in follicular lymphoma (FL) impacts its clinical course, but the interaction between FL cells and host immune cells is poorly understood, and may be influenced by large genetic abnormalities in FL cells. Regions of copy number variation (CNV) and copy-neutral loss of heterozygosity (cnLOH) are detectable by single nucleotide polymorphism (SNP) arrays and frequently found in FL, but the critical “driver genes” within them are largely unidentified. Methods Cell suspensions from tumor biopsies of 66 untreated FL patients were sorted into “B cell” and non-B fractions by immunomagnetic depletion targeting CD3 or CD19 and CD20 respectively. High-resolution Illumina Omni5 SNP arrays were used to profile genomic DNA from B cell fractions and germline DNA (from non-B fractions or peripheral blood cells). Nexus Copy Number software (BioDiscovery) compared paired profiles to determine tumor-specific CNV and cnLOH abnormalities of each patient. Genes within overlapping recurrently-altered regions were identified by the JISTIC algorithm (PMID: 20398270). For 43 of these patients, whole-genome gene expression profiling (GEP) of both fractions was done on Illumina HT12v4 arrays. CONEXIC module network analysis (PMID: 21129771) identified candidate driver genes, based on correlation of their expression in B-cell fractions with that of modules of genes in B-cell or non-B fractions. Results Comparing tumor vs. germline profiles in SNP array analysis clarified the detection of tumor-specific CNV, and enabled the detection of cnLOH. The aggregate genomic profile of regions affected by CNV in our 66 FL samples was highly similar to results of previous FL studies. Most frequent (each in 25-35% of samples) were deletions of 1p36 or a large part of 6q, amplifications of 1q, 7p/q, 12q, 17q, or 18p/q, and cnLOH at 16p. The distribution of these abnormalities suggested that FL can be divided into subgroups based on several large mutually-exclusive genomic aberrations: -10q, -16p, +12q, and, less clearly, -1p/1q+. Novel analysis combining copy number values with corresponding SNP frequencies also identified abnormalities of lower frequency within samples, suggestive of tumor subclones with potential growth advantages, notably including deletions at 13q14 and 19p12 and amplification of 16p13. JISTIC identified 715 expressed genes within amplified regions and 413 expressed genes within deleted regions (329 genes) or regions of cnLOH (84 genes). CONEXIC identified 62 and 68 of these genes as candidate drivers regulating expression of gene modules in tumor B cells and infiltrating immune cells, respectively. Several regulators of B-cell modules were already described in FL or other hematological malignancies: MDM2 (12q15, amplified in 26%), an E3 ubiquitin ligase whose targets include TP53; NME1 (17q21.33, amplified in 21%), part of the nucleoside diphosphate kinase complex, overexpressed and correlated with poor prognosis in AML; or B-cell receptor-associated CD79B (17q23.3, amplified in 21%), mutated and functionally significant in diffuse large B-cell lymphoma. Validating MDM2 as a driver gene, Gene Set Enrichment Analysis showed strong positive association between expression of MDM2 and that of proliferation signatures in B cells, including signatures of genes downregulated by TP53. Genes affecting the interaction between tumor B cells and the FL microenvironment plausibly regulate module expression in both B cells and non-B cells. Such dual candidate driver genes included PHIP (6q14.1, deleted in 27%), a binding partner of insulin receptor substrate-1, overexpressed in melanoma and linked to its metastasis and progression; SMARCC2 (12q13.2, amplified in 25%), part of the ATP-dependent chromatin remodeling complex SNF/SWI, mutated in some carcinomas; SFR1 (10q25.1, deleted in 18%), involved in DNA homologous recombination; and BUD31(7q22.1, amplified in 21%), a homolog of a yeast protein involved in pre-mRNA splicing. Conclusions CNV and cnLOH abnormalities are frequent in FL, and may identify subgroups within FL. Integrated analysis finds known candidate driver genes within recurrently-altered regions, appearing to regulate expression of gene modules in B cells. Novel candidate driver genes that appear to regulate modules in both B and non-B cells may shape the FL microenvironment in important ways, and are being investigated experimentally. Disclosures: No relevant conflicts of interest to declare.

Integrated Analysis of High-Resolution Gene Expression and Copy Number Profiling Identified Biallelic Deletion of CDKN2A/2B Tumor Suppressor Locus As the Most Frequent and Unique Genomic Abnormality in Diffuse Large B-Cell Lymphoma (DLBCL) with Strong Prognostic Value in Both GCB and ABC Subtypes and Not Overcome by a Dose-Intensive Immunochemotherapy Regimen Plus Rituximab. Results of a Prospective GELA Clinical Trial Program

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 415-415 ◽  
Author(s):  
Fabrice Jardin ◽  
Sylvain Mareschal ◽  
Martin Figeac ◽  
Jean-Philippe Jais ◽  
Karen Leroy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
High Resolution ◽  
Tumor Suppressor ◽  
B Cell ◽  
Copy Number ◽  
Gene Copy ◽  
Integrated Analysis ◽  
Driver Genes ◽  
Clinical Trial Program

Abstract Abstract 415 Background and aim of the study Genomic gains and losses play a crucial role in the development and progression of DLBCL and are closely related to gene expression profiles (GEP), including the germinal center B-cell like (GCB) and activated B-cell like (ABC) cell of origin (COO) molecular signatures. To identify new oncogenes or tumor suppressor genes (TSG) involved in DLBCL pathogenesis and to determine their prognostic values, an integrated analysis of high-resolution gene expression and copy number profiling was performed. Patients and methods Two hundred and eight adult patients with de novo CD20+ DLBCL enrolled in the prospective multicentric randomized LNH-03 GELA trials (LNH03-1B, -2B, -3B, 39B, -5B, -6B, -7B) with available frozen tumour samples, centralized reviewing and adequate DNA/RNA quality were selected. 116 patients were treated by Rituximab(R)-CHOP/R-miniCHOP and 92 patients were treated by the high dose (R)-ACVBP regimen dedicated to patients younger than 60 years (y) in frontline. Tumour samples were simultaneously analysed by high resolution comparative genomic hybridization (CGH, Agilent, 144K) and gene expression arrays (Affymetrix, U133+2). Minimal common regions (MCR), as defined by segments that affect the same chromosomal region in different cases, were delineated. Gene expression and MCR data sets were merged using Gene expression and dosage integrator algorithm (GEDI, Lenz et al. PNAS 2008) to identify new potential driver genes. Results A total of 1363 recurrent (defined by a penetrance > 5%) MCRs within the DLBCL data set, ranging in size from 386 bp, affecting a single gene, to more than 24 Mb were identified by CGH. Of these MCRs, 756 (55%) showed a significant association with gene expression: 396 (59%) gains, 354 (52%) single-copy deletions, and 6 (67%) homozygous deletions. By this integrated approach, in addition to previously reported genes (CDKN2A/2B, PTEN, DLEU2, TNFAIP3, B2M, CD58, TNFRSF14, FOXP1, REL…), several genes targeted by gene copy abnormalities with a dosage effect and potential physiopathological impact were identified, including genes with TSG activity involved in cell cycle (HACE1, CDKN2C) immune response (CD68, CD177, CD70, TNFSF9, IRAK2), DNA integrity (XRCC2, BRCA1, NCOR1, NF1, FHIT) or oncogenic functions (CD79b, PTPRT, MALT1, AUTS2, MCL1, PTTG1…) with distinct distribution according to COO signature. The CDKN2A/2B tumor suppressor locus (9p21) was deleted homozygously in 27% of cases and hemizygously in 9% of cases. Biallelic loss was observed in 49% of ABC DLBCL and in 10% of GCB DLBCL. This deletion was strongly correlated to age and associated to a limited number of additional genetic abnormalities including trisomy 3, 18 and short gains/losses of Chr. 1, 2, 19 regions (FDR < 0.01), allowing to identify genes that may have synergistic effects with CDKN2A/2B inactivation. With a median follow-up of 42.9 months, only CDKN2A/2B biallelic deletion strongly correlates (FDR p.value < 0.01) to a poor outcome in the entire cohort (4y PFS = 44% [32–61] respectively vs. 74% [66–82] for patients in germline configuration; 4y OS = 53% [39–72] vs 83% [76–90]). In a Cox proportional hazard prediction of the PFS, CDKN2A/2B deletion remains predictive (HR = 1.9 [1.1–3.2], p = 0.02) when combined with IPI (HR = 2.4 [1.4–4.1], p = 0.001) and GCB status (HR = 1.3 [0.8–2.3], p = 0.31). This difference remains predictive in the subgroup of patients treated by R-CHOP (4y PFS = 43% [29–63] vs. 66% [55–78], p=0.02), in patients treated by R-ACVBP (4y PFS = 49% [28–84] vs. 83% [74–92], p=0.003), and in GCB (4y PFS = 50% [27–93] vs. 81% [73–90], p=0.02), or ABC/unclassified (5y PFS = 42% [28–61] vs. 67% [55–82] p = 0.009) molecular subtypes (Figure 1). Conclusion We report for the first time an integrated genetic analysis of a large cohort of DLBCL patients included in a prospective multicentric clinical trial program allowing identifying new potential driver genes with pathogenic impact. However CDKN2A/2B deletion constitutes the strongest and unique prognostic factor of chemoresistance to R-CHOP, regardless the COO signature, which is not overcome by a more intensified immunochemotherapy. Patients displaying this frequent genomic abnormality warrant new and dedicated therapeutic approaches. Disclosures: Salles: roche: Consultancy.

Gene Expression Signature of Host Immune Response Is Predictive of Follicular Lymphoma Patient Survival in Independent Cohorts, and Correlates with Transformation to Diffuse Large B-Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2951-2951
Author(s):  
Ash A Alizadeh ◽  
Andrew J Gentles ◽  
Sylvia K Plevritis ◽  
Ronald Levy
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Immune Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Independent Cohort

Abstract Abstract 2951 Poster Board II-927 Background: Expression signatures of infiltrating immune cells [1] have been shown to predict survival in follicular lymphoma (FL), but have not been cross-validated in independent patient cohorts [2,3]. These signatures may relate biologically to the frequency of infiltrating including T-cells and macrophages, or to specific transcription programs within tumor cells and/or the tumor microenvironment. We sought to evaluate the validity of this model in an independent cohort of patients with FL, assessing its relationship to outcomes including histological transformation and death. Methods: The immune response (IR) predictor score proposed by Dave et al. [1] was applied to gene expression data from an independent cohort of 88 FL patients [4] with known survival outcomes and history of transformation to diffuse large B-cell lymphoma (DLBCL). Genes (n=66) corresponding to IR1 and IR2 signatures were mapped from Affymetrix microarrays [1] to a custom cDNA array [4] via Entrez Gene ID, and the composite IR score was calculated per the scheme proposed by Dave et al. Results: The IR score was predictive of patient outcome in the 88 patient test set as a continuous variable (p=0.001, HR=2.01, 95% CI 0.50-1.30). Partitioning of patients into high and low risk groups based on the median IR score across the cohort robustly separated survival curves (Figure A). The IR score was significantly higher in FL patients known to undergo transformation to DLBCL (Figure B: mean IR score of -0.6 in non-transforming FL vs. -0.2 in transforming FL; p∼10-11, t-test). Conclusions: The IR score of Dave et al. was highly significant as a predictor of survival in the independent patient cohort [4]. Moreover, the score was significantly associated with propensity of FL to transform to DLBCL. To our knowledge, immune cell infiltration has not previously been implicated in transformation. 1. Dave SS et al. (2004) Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N Engl J Med 351(21): 2159-2169. 2. Tibshirani R (2005) Immune signatures in follicular lymphoma. N Engl J Med 352: 1496-1497. 3. Chu G Hong WJ, Warnke R, Chu G (2005). Immune Signatures in Follicular Lymphoma (Corres). N Engl J Med. 352: 1496-1497. 4. Glas AM et al. (2005) Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment. Blood 105(1): 301-307. Disclosures: No relevant conflicts of interest to declare.

Examining the Heterogeneity of Follicular Lymphoma By Multi-Parameter Flow Cyotmetry in Previously Untreated Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2947-2947
Author(s):  
Debra K Czerwinski ◽  
Steven R Long ◽  
Michael Khodadoust ◽  
Matthew J. Frank ◽  
Adel Kardosh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Tumor Cells ◽  
Immune Cells ◽  
Research Funding ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

Abstract BACKGROUND: Follicular lymphoma (FL) is an indolent form of Non-Hodgkin B cell lymphoma that remains incurable with present therapies. Derived from germinal center B cells, FL B cells experience ongoing hypermutation of the immunoglobulin variable region gene. In addition, Michael Green, et al (PNAS; 2015), reported the presence of numerous somatic mutations to include those of the chromatin-modifying genes. These mutations accumulate over the course of the disease and play an important role in regulating gene transcription, B cell development and immune interactions. Furthermore, FL tumors maintain a resemblance to primary lymphoid follicles, and as such, present with a number of infiltrating immune cells, especially T cells, the numbers of which vary from patient to patient. The close association and interaction of these immune cells with the tumor B cells play an important part in determining the disease biology (Dave SS, et al. N Engl J Med; 2004). For instance, tumor B cells, through cell-cell contact with these immune cells and/or through secretion of inhibitory cytokines such as TGF-b and IL-10, induce T cell exhaustion and apoptosis as well as suppressive T cell phenotypes (FoxP3+ T Regulatory cells) thus evading immune eradication (Yang Z-Z, et al. Blood 2007 and Ai WZ, et al. IntJ Cancer; 2009). They also promote their own survival and proliferation through their interaction with resident T follicular helper cells via CD40L/CD40 interactions (Ame'-Thomas P, et al. Blood; 2005). As a corollary to an ongoing clinical trial, we received fine needle aspirates (FNAs) of easily accessible tumors from 14 patients with FL prior to any treatment. 6 of these patients had samples taken from a second site simultaneously. All samples were processed within 24 hours into a single-cell suspension; red blood cells were lysed. Cells were then stained with antibodies to delineate T, B, NK, dendritic, and myeloid cells, as well as their subsets. Antibodies against activation antigens, T cell exhaustion, inhibition and function were also used to characterize these cells. Finally, the cells were run on a 17-parameter LSRII (Becton Dickinson) and data analyzed via Cytobank, a web-based data storage and analysis tool. PURPOSE: To better understand the biology of FL as represented by protein expression by the tumor cells and the immune cells that make up the microenvironment. We will especially look to evaluate the heterogeneity inherent in FL by flow cytometry across patients as well as within any one individual. RESULTS: Each sample is stained with 4 panels of antibodies, 13 antibodies each, allowing us to measure over 100 cell subsets. A quick preview of all data shows that there is a high variability between patients in the percentage of T cells within the microenvironment (37.7% + 16.6% of all cells collected from all samples). This variability is represented by the differences in the CD4 T cell compartment (27.6 + 12.9%) and to a lesser degree in the CD8 compartment (7.7 + 3.7%). To note, this variability in T cells does not correlate with time from diagnosis to sample collection which ranged from 3.4 years to approximately 5 months. Also, this is in contrast to the similar percentage of CD4 and CD8 T cells expressing PD-1 (55.5 + 8.8% and 46.0 + 8.9%, respectively) across patients. Notably, there is much less variability from site to site within each patient then between patients as demonstrated by Figure 1 where Site A and Site B are 2 separate lesions within each patient listed, sampled at the same time. Since FL presumably begins in a single site in the body and then becomes disseminated, the fact that a characteristic relationship exists between tumor cells and immune cells wherever the disease is found implies a mutual interdependence of the tumor cells in each case and their immune host component. CONCLUSION: Follicular lymphoma is a very heterogeneous disease as would be expected by the diversity of mutations seen at the genomic level. This heterogeneity is also apparent in the microenvironment from one patient to another. Conversely, different tumor sites within each patient have a characteristic and fixed relationship to their immune microenvironment. The emergence of novel therapies for FL, including checkpoint antibodies such as anti-PD-1 and anti-PD-L1 and small molecules such as Ibrutinib, will be informed by understanding the differences as well as the similarities in each case of FL. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

scholarly journals A dichotomy of gene regulatory associations during the activated B-cell to plasmablast transition

2020 ◽  
Vol 3 (10) ◽  
pp. e202000654
Author(s):  
Mario Cocco ◽  
Matthew A Care ◽  
Amel Saadi ◽  
Muna Al-Maskari ◽  
Gina Doody ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Integrated Analysis ◽  
Plasma Cell Myeloma ◽  
Signal Loss ◽  
Secreting Cell ◽  
Antibody Secreting Cell ◽  
Er Stress Response ◽  
Gene Modules ◽  
Gene Regulatory

The activated B-cell (ABC) to plasmablast transition encompasses the cusp of antibody-secreting cell (ASC) differentiation. We explore this transition with integrated analysis in human cells, focusing on changes that follow removal from CD40-mediated signals. Within hours of input signal loss, cell growth programs shift toward enhanced proliferation, accompanied by ER-stress response, and up-regulation of ASC features. Clustering of genomic occupancy for IRF4, BLIMP1, XBP1, and CTCF with histone marks identifies a dichotomy: XBP1 and IRF4 link to induced but not repressed gene modules in plasmablasts, whereas BLIMP1 links to modules of ABC genes that are repressed, but not to activated genes. Between ABC and plasmablast states, IRF4 shifts away from AP1/IRF composite elements while maintaining occupancy at IRF and ETS/IRF elements. This parallels the loss of BATF expression, which is identified as a potential BLIMP1 target. In plasmablasts, IRF4 acquires an association with CTCF, a feature maintained in plasma cell myeloma lines. Thus, shifting occupancy links IRF4 to both ABC and ASC gene expression, whereas BLIMP1 occupancy links to repression of the activation state.

Characterization Of The Effects Of Mutated EZH2 On Expression and Epigenome In a Mouse Lymphoma Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 346-346
Author(s):  
Tobias Berg ◽  
Silvia Thoene ◽  
Damian Yap ◽  
Tracee Wee ◽  
Nathalie Schoeler ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Rna Sequencing ◽  
Transcription Start Site ◽  
Integrated Analysis ◽  
Start Site ◽  
Sequencing Data ◽  
Transcription Start ◽  
Chip Sequencing

Abstract The histone methyl-transferase EZH2 is frequently affected by gain-of-function mutations in germinal-center lymphoma. To further test if these EZH2 mutations can be driver mutations in lymphomagenesis, we have previously generated and characterized a mouse line transgenic for EZH2Y641F. This mouse model leads to an acceleration of lymphoma development in combination with Myc. We have now further investigated this model system and have used RNAseq and ChIPseq to characterize the effects of mutated EZH2 on gene expression and the epigenome. We have previously shown that in contrast to lymphomas observed with Emu-Myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2Y641F, present with a B220+IgM+phenotype. We have now characterized the accumulating B cell subset by performing in-depth immunophenotyping. This analysis showed that the cells are IgDlo, CD21-, CD23- and partially express AA4.1. This marker combination is consistent with the transitional stage (T1) of B cell development. For the global gene expression and histone methylation analysis, splenic B cells (B220+) were isolated from one Eμ-Myc and one Eμ-Myc/EZH2Y641F mouse before the onset of disease symptoms. The samples were then processed for RNA sequencing and ChIP sequencing. ChIP was performed using validated antibodies for H3K4me3 and H3K27me3. Of the 22,137 genes studied we observed that 1,112 (412) genes were down-regulated and 788 (209) genes were up-regulated in Eμ-Myc/EZH2Y641F mice with p-value<0.05 (FDR<0.1) when only genes with RPKM > 0.05 and a minimum number of reads of 30 were considered. An integrated analysis of the RNA sequencing data with the ChIP sequencing data for H3K27me3 and H3K4me3 was performed, and indicated that the altered epigenome of the Eμ-Myc/EZH2Y641F mouse impacted protein-coding gene expression. This analysis showed that genes down-regulated in the Eμ-Myc/EZH2Y641F mouse have increased H3K27me3 marks at their transcription start site indicative of a significant fraction of these genes being regulated by this mark. In contrast, genes up-regulated in the Eμ-Myc/EZH2Y641Fmouse mainly exhibited increased H3K4Me3 marks at their transcription start site. We then used DAVID/KEGG and the MGSA package to identify pathways associated with mutated EZH2 and identified GO terms that were enriched by the differentially expressed genes. Interestingly this analysis returned many important pathways in B cell regulation and immune function. The identified pathways include the B cell receptor signalling pathway and the JAK-STAT signaling pathway. We previously observed an increase in the proliferation rate of splenic B cells in Eμ-Myc/EZH2Y641Fmice. While cell-cycle genes were not specifically enriched, there were several of these genes that were found deregulated (including e. g. CyclinD1). In summary, we were able to identify several key pathways that may be contributing to the acceleration of lymphoma development observed with EZH2Y641F and may also be important for the understanding of pathogenesis of EZH2 mutated lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single-cell RNA-Seq of follicular lymphoma reveals malignant B-cell types and coexpression of T-cell immune checkpoints

Blood ◽  
2019 ◽  
Vol 133 (10) ◽  
pp. 1119-1129 ◽  
Author(s):  
Noemi Andor ◽  
Erin F. Simonds ◽  
Debra K. Czerwinski ◽  
Jiamin Chen ◽  
Susan M. Grimes ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
Immune Checkpoint ◽  
Gene Networks ◽  
Immune Checkpoints ◽  
Transcriptional Networks ◽  
Low Grade

Abstract Follicular lymphoma (FL) is a low-grade B-cell malignancy that transforms into a highly aggressive and lethal disease at a rate of 2% per year. Perfect isolation of the malignant B-cell population from a surgical biopsy is a significant challenge, masking important FL biology, such as immune checkpoint coexpression patterns. To resolve the underlying transcriptional networks of follicular B-cell lymphomas, we analyzed the transcriptomes of 34 188 cells derived from 6 primary FL tumors. For each tumor, we identified normal immune subpopulations and malignant B cells, based on gene expression. We used multicolor flow cytometry analysis of the same tumors to confirm our assignments of cellular lineages and validate our predictions of expressed proteins. Comparison of gene expression between matched malignant and normal B cells from the same patient revealed tumor-specific features. Malignant B cells exhibited restricted immunoglobulin (Ig) light chain expression (either Igκ or Igλ), as well the expected upregulation of the BCL2 gene, but also downregulation of the FCER2, CD52, and major histocompatibility complex class II genes. By analyzing thousands of individual cells per patient tumor, we identified the mosaic of malignant B-cell subclones that coexist within a FL and examined the characteristics of tumor-infiltrating T cells. We identified genes coexpressed with immune checkpoint molecules, such as CEBPA and B2M in regulatory T (Treg) cells, providing a better understanding of the gene networks involved in immune regulation. In summary, parallel measurement of single-cell expression in thousands of tumor cells and tumor-infiltrating lymphocytes can be used to obtain a systems-level view of the tumor microenvironment and identify new avenues for therapeutic development.

scholarly journals High-Throughput Molecular Profiling of Multiple Myeloma (MM) Clonotypic CD19+ B Cells Highlights Pathways Potentially Involved in the Disease Endurance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2054-2054
Author(s):  
Marina Martello ◽  
Angela Flores Dico ◽  
Daniel Remondini ◽  
Giovanni Martinelli ◽  
Enrica Borsi ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Molecular Characterization ◽  
Gene Rearrangement ◽  
Memory B Cells ◽  
Newly Diagnosed ◽  
Cell Compartment ◽  
Cell Fractions ◽  
Immature Cells

Abstract Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics. Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence. Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively. Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed. SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A). Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (<-2FC>2; FDR=0,05; p <0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed in every analyzed cell compartment; on the contrary, Hedgehog pathway was overall down regulated. Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p=7,25E-14; FDR= 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC=-18,0; -19,96. p<0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells. Conclusions Presented data support the emerging role of the immature cell compartment in the MM disease course, where the MM CD138+ clone might resume the end of the complex process of tumorigenesis, whereas the putative CD19+ MPCs, by displaying peculiar genomic micro-alterations and a unique transcriptional profile, might be involved in the neoplastic clone supply. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy. Cavo:BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

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.

scholarly journals Antibody repertoire and gene expression dynamics of diverse human B cell states during affinity maturation

2020 ◽  
Author(s):  
Hamish W King ◽  
Nara Orban ◽  
John C Riches ◽  
Andrew J Clear ◽  
Gary Warnes ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Affinity Maturation ◽  
Germinal Centre ◽  
Integrated Analysis ◽  
Cell Mediated Immunity ◽  
Antibody Repertoires ◽  
B Cell Subsets

AbstractIn response to antigen challenge, B cells clonally expand, undergo selection and differentiate to produce mature B cell subsets and high affinity antibodies. However, the interplay between dynamic B cell states and their antibody-based selection is challenging to decipher in primary human tissue. We have applied an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics of human B cells undergoing affinity maturation. We define unique gene expression and antibody repertoires of known and novel B cell states, including a pre-germinal centre state primed to undergo class switch recombination. We dissect antibody class-dependent gene expression of germinal centre and memory B cells to find that class switching prior to germinal centre entry dictates the capacity of B cells to undergo antibody-based selection and differentiate. Together, our analyses provide unprecedented resolution into the gene expression and selection dynamics that shape B cell-mediated immunity.

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