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Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.
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Linfoma Difuso de Grandes Células B , NF-kappa B , Transdução de Sinais , Fator 3 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Humanos , NF-kappa B/metabolismo , Quinase Induzida por NF-kappaB , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proliferação de CélulasRESUMO
Follicular lymphoma (FL) accounts for â¼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.
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Linfoma Folicular , Linfoma Difuso de Grandes Células B , Humanos , Linfoma Folicular/patologia , Mutação , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Long non-coding RNAs (lncRNAs) are involved in many normal and oncogenic pathways through a diverse repertoire of transcriptional and posttranscriptional regulatory mechanisms. LncRNAs that are under tight regulation of well-known oncogenic transcription factors such as c-Myc (Myc) are likely to be functionally involved in their disease-promoting mechanisms. Myc is a major driver of many subsets of B cell lymphoma and to date remains an undruggable target. We identified three Myc-induced and four Myc-repressed lncRNAs by use of multiple in vitro models of Myc-driven Burkitt lymphoma and detailed analysis of Myc binding profiles. We show that the top Myc-induced lncRNA KTN1-AS1 is strongly upregulated in different types of B cell lymphoma compared with their normal counterparts. We used CRISPR-mediated genome editing to confirm that the direct induction of KTN1-AS1 by Myc is dependent on the presence of a Myc E-box-binding motif. Knockdown of KTN1-AS1 revealed a strong negative effect on the growth of three BL cell lines. Global gene expression analysis upon KTN1-AS1 depletion shows a strong enrichment of key genes in the cholesterol biosynthesis pathway as well as co-regulation of many Myc-target genes, including a moderate negative effect on the levels of Myc itself. Our study suggests a critical role for KTN1-AS1 in supporting BL cell growth by mediating co-regulation of a variety of Myc-target genes and co-activating key genes involved in cholesterol biosynthesis. Therefore, KTN1-AS1 may represent a putative novel therapeutic target in lymphoma.
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Linfoma de Burkitt , Linfoma de Células B , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Proliferação de Células/genética , Colesterol , Proteínas de Membrana/genéticaRESUMO
INTRODUCTION: The current obesity crisis has resulted in many people with excess adipose tissue suffering from chronic inflammation. This inflammation is largely due to the release of cytokines and chemokines from visceral fat. The aim of this study was to identify potential anti-inflammatory agents that might alleviate obesity-induced chronic inflammation. METHODS: To identify agents that might alleviate this obesity-induced chronic inflammation we have developed a simple protocol for incubating intact pieces of human visceral adipose tissue in 35 mm tissue culture plates, in the presence of low-dose lipopolysaccharide (LPS) and co-incubating these samples with potential anti-inflammatory agents. RNA-Seq analysis was performed to identify enriched gene expression signatures among the most significantly differentially expressed genes. RESULTS: From this screen, we have identified the short-chain fatty acid (SCFA) sodium butyrate and its triacylglyceride form, tributyrin, as effective agents, significantly reducing the production of LPS-induced inflammatory cytokines and chemokines from all adipose tissue samples tested. As well, these agents appear to be non-toxic at the concentrations tested. RNA-Seq analysis has revealed that IL36γ is one of the most upregulated genes in response to LPS and one of the most downregulated when sodium butyrate is added to human fat samples stimulated with LPS. IL-36γ ELISAs confirmed this holds true at the protein level as well. CONCLUSIONS: These studies suggest that the short-chain fatty acid, sodium butyrate, and its triacylglyceride form, tributyrin, might alleviate the chronic inflammation that is associated with many individuals with obesity.
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BACKGROUND: Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. METHODS: Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. RESULTS: We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). CONCLUSIONS: A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.
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Leucemia Linfocítica Crônica de Células B , Hipermutação Somática de Imunoglobulina , Humanos , Alelos , Sequenciamento de Nucleotídeos em Larga Escala , Imunoglobulinas , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Fatores de TranscriçãoRESUMO
Primary mediastinal large B-cell lymphoma (PMBL) is a type of aggressive B-cell lymphoma that typically affects young adults, characterized by presence of a bulky anterior mediastinal mass. Lymphomas with gene expression features of PMBL have been described in nonmediastinal sites, raising questions about how these tumors should be classified. Here, we investigated whether these nonmediastinal lymphomas are indeed PMBLs or instead represent a distinct group within diffuse large B-cell lymphoma (DLBCL). From a cohort of 325 de novo DLBCL cases, we identified tumors from patients without evidence of anterior mediastinal involvement that expressed a PMBL expression signature (nm-PMBLsig+; n = 16; 5%). A majority of these tumors expressed MAL and CD23, proteins typically observed in bona fide PMBL (bf-PMBL). Evaluation of clinical features of nm-PMBLsig+ cases revealed close associations with DLBCL, and a majority displayed a germinal center B cell-like cell of origin (GCB). In contrast to patients with bf-PMBL, patients with nm-PMBLsig+ presented at an older age and did not show pleural disease, and bone/bone marrow involvement was observed in 3 cases. However, although clinically distinct from bf-PMBL, nm-PMBLsig+ tumors resembled bf-PMBL at the molecular level, with upregulation of immune response, JAK-STAT, and NF-κB signatures. Mutational analysis revealed frequent somatic gene mutations in SOCS1, IL4R, ITPKB, and STAT6, as well as CD83 and BIRC3, with the latter genes significantly more frequently affected than in GCB DLBCL or bf-PMBL. Our data establish nm-PMBLsig+ lymphomas as a group within DLBCL with distinct phenotypic and genetic features. These findings may have implications for gene expression- and mutation-based subtyping of aggressive B-cell lymphomas and related targeted therapies.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Neoplasias do Mediastino/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Feminino , Células HEK293 , Humanos , Evasão da Resposta Imune , Imunofenotipagem , Janus Quinases/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Masculino , Neoplasias do Mediastino/patologia , Pessoa de Meia-Idade , Mutação/genética , Receptores de Interleucina-4/genética , Fatores de Transcrição STAT/metabolismo , Hipermutação Somática de Imunoglobulina/genética , Adulto JovemRESUMO
The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, were analyzed as a discovery cohort, followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) exhibited a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (n = 18) had a significantly distinct pattern that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV- GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas.
Assuntos
Doença de Hodgkin/genética , Linfoma Difuso de Grandes Células B/genética , Neoplasias do Mediastino/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Feminino , Doença de Hodgkin/complicações , Humanos , Linfoma Difuso de Grandes Células B/complicações , Masculino , Neoplasias do Mediastino/complicações , Pessoa de Meia-Idade , Timo/metabolismo , Adulto JovemAssuntos
Linfoma Difuso de Grandes Células B , RNA Longo não Codificante , Humanos , Transcriptoma , RNA Longo não Codificante/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Perfilação da Expressão Gênica , Resultado do TratamentoRESUMO
Despite numerous studies reporting deregulated microRNA (miRNA) and gene expression patterns in clear cell renal cell carcinoma (ccRCC), no direct comparisons have been made to its presumed normal counterpart: the renal proximal tubular epithelial cells (PTECs). The aim of this study was to determine the miRNA expression profiles of 10 ccRCC-derived cell lines and short-term cultures of PTEC and to correlate these with their gene expression and copy-number profiles. Using microarray-based methods, a significantly altered expression level in ccRCC cell lines was observed for 23 miRNAs and 1630 genes. The set of miRNAs with significantly decreased expression levels include all members of the miR-200 family known to be involved in the epithelial to mesenchymal transition process. Expression levels of 13 of the 47 validated target genes for the downregulated miRNAs were increased more than twofold. Our data reinforce the importance of the epithelial to mesenchymal transition process in the development of ccRCC.
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Carcinoma de Células Renais/genética , Células Epiteliais/metabolismo , Neoplasias Renais/genética , Túbulos Renais Proximais/metabolismo , MicroRNAs/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Túbulos Renais Proximais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Vimentina/genética , Vimentina/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
PURPOSE: A genetic classifier termed LymphGen accurately identifies diffuse large B-cell lymphoma (DLBCL) subtypes vulnerable to Bruton's tyrosine kinase inhibitors (BTKis), but is challenging to implement in the clinic and fails to capture all DLBCLs that benefit from BTKi-based therapy. Here, we developed a novel CD5 gene expression signature as a biomarker of response to BTKi-based therapy in DLBCL. METHODS: CD5 immunohistochemistry (IHC) was performed on 404 DLBCLs to identify CD5 IHC+ and CD5 IHC- cases, which were subsequently characterized at the molecular level through mutational and transcriptional analyses. A 60-gene CD5 gene expression signature (CD5sig) was constructed using genes differentially expressed between CD5 IHC+ and CD5 IHC- non-germinal center B-cell-like (non-GCB DLBCL) DLBCLs. This CD5sig was applied to external DLBCL data sets, including pretreatment biopsies from patients enrolled in the PHOENIX study (n = 584) to define the extent to which the CD5sig could identify non-GCB DLBCLs that benefited from the addition of ibrutinib to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: CD5 expression was observed in 12% of non-GCB DLBCLs. CD5+ DLBCLs displayed transcriptional features of B-cell receptor (BCR) activation and were enriched for BCR-activating mutations known to correlate with BTKi sensitivity. However, most CD5+ DLBCLs lacked canonical BCR-activating mutations or were LymphGen-unclassifiable (LymphGen-Other). The CD5sig recapitulated these findings in multiple independent data sets, indicating its utility in identifying DLBCLs with genetic and nongenetic bases for BCR dependence. Supporting this notion, CD5sig+ DLBCLs derived a selective survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study, independent of LymphGen classification. CONCLUSION: CD5sig is a useful biomarker to identify DLBCLs vulnerable to BTKi-based therapies and complements current biomarker approaches by identifying DLBCLs with genetic and nongenetic bases for BTKi sensitivity.
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Linfoma Difuso de Grandes Células B , Humanos , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfócitos B/patologia , Rituximab/uso terapêutico , Vincristina/uso terapêutico , Biomarcadores , Doxorrubicina/uso terapêutico , Ciclofosfamida/uso terapêutico , Prednisona/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , PrognósticoRESUMO
Most diffuse large B-cell lymphoma (DLBCL) patients treated with bispecific antibodies (BsAb) or chimeric antigen receptor (CAR) T cells fail to achieve durable treatment responses, underscoring the need for a deeper understanding of mechanisms that regulate the immune environment and response to treatment. Here, an integrative, multi-omic approach was employed to characterize DLBCL immune environments, which effectively segregated DLBCLs into four quadrants - termed DLBCL-immune quadrants (IQ) - defined by cell-of-origin and immune-related gene set expression scores. Recurrent genomic alterations were enriched in each IQ, suggesting that lymphoma cell-intrinsic alterations contribute to orchestrating unique DLBCL immune environments. In relapsed/refractory DLBCL patients, DLBCL-IQ assignment correlated significantly with clinical benefit with the CD20 x CD3 BsAb, mosunetuzumab, but not with CD19-directed CAR T cells. DLBCL-IQ provides a new framework to conceptualize the DLBCL immune landscape and uncovers the differential impact of the endogenous immune environment on outcomes to BsAb and CAR T cell treatment.
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Progression or relapse in the central nervous system (CNS) remains a rare but mostly fatal event for patients with diffuse large B-cell lymphoma (DLBCL). In a retrospective analysis of 5189 patients treated within 19 prospective German and French phase 2/3 trials, we identified 159 patients experiencing a CNS event (relapse: 62%, progression: 38%). Intracerebral, meningeal, intraspinal, or combined involvement was reported in 44%, 31%, 3%, and 22% of patients, respectively. 62 of 155 evaluable patients (40%) showed concurrent systemic progression/ relapse. 82% of all CNS events occurred within two years after study inclusion or randomization. 87% of patients showed extranodal involvement outside the CNS. Patients generally had poor outcomes with a median overall survival (OS) of 3.4 months (95% CI 2.9-4.2) and a 2-year OS of 15% (10-22%). Outcomes did not differ depending on the site or time point of CNS events. Patients with isolated CNS events demonstrated significantly better OS (p = 0.023). Twenty-five patients were consolidated with autologous or allogeneic stem cell transplantation and achieved a 3-year OS of 36% (20-66%). This large study including more than 5000 DLBCL patients highlights the unmet medical need to improve the outcome of DLBCL patients suffering from CNS relapse.
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We investigated the clinicopathologic features of 5 follicular lymphomas (FLs) that transformed (tFL) morphologically to diffuse large B-cell lymphomas (DLBCLs) and had a primary mediastinal large B-cell lymphoma (PMBL)-like gene expression profile (tFL-PMBLsig-pos). None of the tFL-PMBLsig-pos cases arose in the mediastinum, all cases tested had a germinal center B-cell phenotype, 20% were CD30+, 60% CD23+, 80% MAL+, 20% CD200+, and 0% CD273/PDL2+. Whole-exome sequencing detected alterations in genes associated with both FL/DLBCL (CREBBP, KMT2C, KMT2D, ARID1A, HIST1 members, and TNFRSF14) and PMBL (JAK-STAT pathway genes, B2M, and CD58). Copy number (CN) analysis detected gains/amplification of REL and STAT6 in 60%, gains of SOCS1 in 40%, and gains of chromosome 16, including IL4R, in 40% of the cases. CN gains/amplification of BCL6 and MYC and loss of TNFRSF14 and TNFAIP3 were identified in 20% of the cases. Three of 5 cases lacked a BCL2 rearrangement. Despite having some features that are less common in DLBCL (MAL and CD23 expression and JAK-STAT activation), these tFL-PMBLsig-pos cases lack the most characteristic CN alteration seen in PMBL (9p24.1 gain/amplification). This cohort expands the biologic heterogeneity of tFL, illustrating a subset with gene expression and some genetic features reminiscent of PMBL, with potential treatment implications that include the use of novel targeted therapies.
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Linfoma Folicular , Linfoma Difuso de Grandes Células B , Transcriptoma , Humanos , Janus Quinases , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Transdução de Sinais , Fatores de Transcrição STAT , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Expressão Gênica/genética , Expressão Gênica/fisiologiaRESUMO
Clear cell renal cell carcinomas are characterized by 3p loss, and by inactivation of Von Hippel Lindau (VHL), a tumorsuppressor gene located at 3p25. Recently, SETD2, located at 3p21, was identified as a new candidate ccRCC tumor-suppressor gene. The combined mutational frequency in ccRCC tumors of VHL and SETD2 suggests that there are still undiscovered tumor-suppressor genes on 3p. We screened all genes on 3p for mutations in 10 primary ccRCC tumors using exome-sequencing. We identified inactivating mutations in VHL, PBRM1, and BAP1. Sequencing of PBRM1 in ccRCC-derived cell lines confirmed its frequent inactivation in ccRCC. PBRM1 encodes for BAF180, the chromatin targeting subunit of the SWI/SNF complex. BAP1 encodes for BRCA1 associated protein-1, involved in histone deubiquitination. Taken together, the accumulating data suggest an important role for aberrant chromatin regulation in ccRCC development.
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Carcinoma de Células Renais/genética , Cromatina/metabolismo , Exoma/genética , Carcinoma de Células Renais/metabolismo , Cromatina/genética , Cromossomos Humanos Par 3/genética , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.
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Antígenos de Neoplasias , Linfoma Difuso de Grandes Células B , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/terapia , Microambiente Tumoral/genéticaRESUMO
Follicular lymphoma (FL) is an indolent cancer of mature B-cells but with ongoing risk of transformation to more aggressive histology over time. Recurrent mutations associated with transformation have been identified; however, prognostic features that can be discerned at diagnosis could be clinically useful. We present here comprehensive profiling of both tumor and immune compartments in 155 diagnostic FL biopsies at single-cell resolution by mass cytometry. This revealed a diversity of phenotypes but included two recurrent patterns, one which closely resembles germinal center B-cells (GCB) and another which appears more related to memory B-cells (MB). GCB-type tumors are enriched for EZH2, TNFRSF14, and MEF2B mutations, while MB-type tumors contain increased follicular helper T-cells. MB-type and intratumoral phenotypic diversity are independently associated with increased risk of transformation, supporting biological relevance of these features. Notably, a reduced 26-marker panel retains sufficient information to allow phenotypic profiling of future cohorts by conventional flow cytometry.
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Linfoma Folicular , Humanos , Linfoma Folicular/genética , Células B de Memória , Centro Germinativo , Linfócitos B , MutaçãoRESUMO
The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that approximately 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.
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Regulação da Expressão Gênica , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Argonautas , Sítios de Ligação , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/isolamento & purificação , Fator de Iniciação 2 em Eucariotos/metabolismo , Genes Reporter , Humanos , Imunoprecipitação , Luciferases/genética , Reprodutibilidade dos TestesRESUMO
As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.
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Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Estudos de Coortes , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Feminino , Fusão Gênica , Humanos , Mutação INDEL , Integrinas/genética , Integrinas/metabolismo , Leucemia Mieloide Aguda/genética , Masculino , Polimorfismo de Nucleotídeo Único , Prognóstico , Estudos Prospectivos , RNA-Seq , Fatores de Risco , Transdução de Sinais/genética , Análise de Sobrevida , Transcriptoma , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
Genomic alterations in cancer cells can influence the immune system to favor tumor growth. In non-Hodgkin lymphoma, physiological interactions between B cells and the germinal center microenvironment are coopted to sustain cancer cell proliferation. We found that follicular lymphoma patients harbor a recurrent hotspot mutation targeting tyrosine 132 (Y132D) in cathepsin S (CTSS) that enhances protein activity. CTSS regulates antigen processing and CD4+ and CD8+ T cell-mediated immune responses. Loss of CTSS activity reduces lymphoma growth by limiting communication with CD4+ T follicular helper cells while inducing antigen diversification and activation of CD8+ T cells. Overall, our results suggest that CTSS inhibition has non-redundant therapeutic potential to enhance anti-tumor immune responses in indolent and aggressive lymphomas.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Catepsinas/genética , Linfoma não Hodgkin/imunologia , Mutação , Microambiente Tumoral/imunologia , Animais , Apoptose , Linfócitos B/imunologia , Proliferação de Células , Feminino , Centro Germinativo/imunologia , Humanos , Ativação Linfocitária/imunologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/imunologia , Células Tumorais CultivadasRESUMO
Gray zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classic Hodgkin lymphoma (cHL), is a rare and poorly defined entity. Alongside GZL, a subset of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) has been described with polymorphic/GZL-like morphology (polymorphic-EBV-L). To fill the important gap in our understanding of the pathogenic process underlying these entities, we performed a gene expression study of a large international cohort of GZL and polymorphic-EBV-L, combined with cHL and primary mediastinal large B-cell lymphoma (PMBCL) cases. In an unsupervised principal component analysis, GZL cases presented with intermediate scores in a spectrum between cHL and PMBCL, whereas polymorphic-EBV-L clustered distinctly. The main biological pathways underlying the GZL spectrum were related to cell cycle, reflecting tumor cell content, and extracellular matrix signatures related to the cellular tumor microenvironment. Differential expression analysis and phenotypic characterization of the tumor microenvironment highlighted the predominance of regulatory macrophages in GZL compared with cHL and PMBCL. Two distinct subtypes of GZL were distinguishable that were phenotypically reminiscent of PMBCL and DLBCL, and we observed an association of PMBCL-type GZL with clinical presentation in the "thymic" anatomic niche. In summary, gene expression profiling (GEP) enabled us to add precision to the GZL spectrum, describe the biological distinction compared with polymorphic-EBV-L, and distinguish cases with and without thymic involvement as 2 subgroups of GZL, namely PMBCL-like and DLBCL-like GZL.