RESUMO
AIMS: T cell large granular lymphocytic leukaemia (T-LGLL) is a rare disorder that may underlie otherwise unexplained cytopenias. The identification of T-LGLL cells in bone marrow biopsies can be a challenge, because a robust immunohistochemistry marker is lacking. The markers currently in use (granzyme B, TIA-1 and CD8) are difficult to interpret or lack specificity. Therefore, we investigated whether immunohistochemistry for thymocyte selection-associated high-mobility group box (TOX), a transcription factor that associates with chronic T cell stimulation, could be a reliable tool for the identification of T-LGLL cells. METHODS AND RESULTS: In this retrospective study, expression of TOX in CD8+ cells in bone marrow biopsies of T-LGLL patients (n = 38) was investigated and compared to bone marrow of controls with reactive T cell lymphocytosis (n = 10). All biopsies were evaluated for TOX staining within the CD8-positive T cell population. The controls were essentially negative for TOX, whereas all T-LGLL cases were positive (median = 80%, range = 10-100%), even when bone marrow involvement was subtle. CONCLUSION: TOX is a highly sensitive marker for the neoplastic cells of T-LGLL and we recommend its use, especially in the diagnostic work-up of patients with unexplained cytopenias.
Assuntos
Leucemia Linfocítica Granular Grande , Linfocitose , Humanos , Medula Óssea/patologia , Linfócitos T CD8-Positivos/patologia , Leucemia Linfocítica Granular Grande/diagnóstico , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Linfocitose/patologia , Estudos RetrospectivosRESUMO
BACKGROUND: Proliferation of large granular lymphocytes (LGL) and T-cell LGL (T-LGL) in peripheral blood along with demonstration of clonality are the hallmarks of a heterogeneous group of disorders, including T-LGL leukemia or T-LGL lymphocytosis. They are often associated with neutropenia and responsive to immunosuppression. The true nature of this entity is not well understood. Some cases are reported as reactive phenomena with very limited experience in pediatric population. METHODS: Hematology/Oncology Flow Cytometry Laboratory database has been reviewed retrospectively. Patients with identifiable distinct CD5-dim T-cell population and positive clonal T-cell receptor rearrangement were included in the analysis. Clinical and laboratory data were then reviewed. RESULTS: Sixteen cases of children and young adults with increased peripheral blood clonal T-LGL population characterized by dim CD5 expression with wide range of underlying immune dysregulation/stimulation disorders were reviewed. Extended follow up with repeat testing suggested the reactive nature of persistent clonal T-LGL proliferations in this group. CONCLUSIONS: Our observations indicate that clonal T-LGL proliferations in children and young adults are reactive in nature and some can be persistent with an indolent course with unknown consequentiality. Clonal T-LGL cells could be targeting the most prominent immunogenic stressor(s) involved as a control mechanism.
Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos CD5/metabolismo , Proliferação de Células , Leucemia Linfocítica Granular Grande , Linfócitos T , Adolescente , Adulto , Criança , Bases de Dados Factuais , Feminino , Seguimentos , Humanos , Lactente , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Masculino , Estudos Retrospectivos , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Calcitriol, the active form of vitamin D, has been well documented to act directly on immune cells and malignant cells. Activated T cells are one of the best characterized targets of calcitriol, with effects including decreasing inflammatory cytokine output and promoting anti-inflammatory cytokine production. However, the effects of calcitriol on natural killer (NK) cells are less clear. Reports suggest that only immature NK cell populations are affected by calcitriol treatment resulting in impaired cytotoxic function and cytokine production, while mature NK cells may have little or no response. NK cell large granular lymphocyte leukemia (NK-LGLL) is a rare leukemia with CD3-CD16+CD56+NK cell clonal expansion. The current standard treatments are immunosuppressant therapies, which are not curative. The Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway is hyperactivated in LGLL and is one pathway of interest in new drug target investigations. We previously demonstrated the ability of calcitriol to decrease STAT1 tyrosine 701 (p-STAT1) and STAT3 tyrosine 705 (p-STAT3) phosphorylation as well as inflammatory cytokine output of T cell large granular lymphocyte leukemia cells, but did not determine the effects of calcitriol on NK-LGLL. Therefore, in the present study, we investigated whether NKL cells, a model of NK-LGLL, and NK-LGLL patient peripheral blood mononuclear cells (PBMCs) are susceptible to treatment with calcitriol or seocalcitol (EB1089), a potent analog of calcitriol. NKL cells are dependent on interleukin (IL)-2 for survival and we show here for the first time that treatment with IL-2 induced tyrosine phosphorylation of STATs 1 through 6. Both calcitriol and EB1089 caused significant upregulation of the vitamin D receptor (VDR). IL-2 induction of p-STAT1 and p-STAT3 phosphorylation was significantly decreased after calcitriol or EB1089 treatment. Additionally, IL-10, interferon (IFN)-γ, and FMS-like tyrosine kinase 3 ligand (Flt-3L) extracellular output was significantly decreased at 100â¯nMâ¯EB1089 and intracellular IL-10 was decreased with either calcitriol or EB1089 treatment. We treated NK-LGLL patient PBMCs with calcitriol or EB1089 and found decreased p-STAT1 and p-STAT3 while VDR increased, which matched the NKL cell line data. We then measured 75 serum cytokines in NK-LGLL patients (nâ¯=â¯8) vs. age- and sex-matched normal healthy donors (nâ¯=â¯8), which is the first serum cytokine study for this LGLL subtype. We identified 15 cytokines, including IL-10 and Flt-3L, which were significantly different between normal donors and NK-LGLL patients. Overall, our results suggest that activating the vitamin D pathway could be a mechanism to decrease STAT1 and 3 activation and inflammatory cytokine output in NK-LGLL patients.
Assuntos
Citocinas/metabolismo , Inflamação/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Vitamina D/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Janus Quinases/metabolismo , Células Matadoras Naturais , Receptores de Calcitriol/metabolismo , Linfócitos T/metabolismoRESUMO
BACKGROUND: Chronic lymphoproliferative disorder of NK-cells (CLPD-NK) manifests as a persistent increase (≥2 × 109 /L, for > 6 months) of mature NK-cells in peripheral blood with an indolent clinical course. The disease is rare, and only limited case series have been published. METHODS: We retrospectively studied 11 patients with CLPD-NK diagnosed at our institution between 2005 and 2017. RESULTS: Patients included 7 men and 4 women with a median age of 60 years (range, 25-89 years). Ten patients (91%) had cytopenias. Bone marrow involvement by CLPD-NK ranged from 5-15%. The most commonly detected antigenic aberrancies by low cytometry immunophenotyping were as follows: CD7decreased/dim (30%), CD8uniform+ (36%), CD56-/partial (73%), CD94bright (55%), and KIR restriction (100%). JAK/STAT pathway mutations were detected in 8 of 10 (80%) patients and involved STAT3 (n = 7) and JAK3 (n = 1). The presence of mutations tended to correlate with the occurrence of other cytopenias (anemia/thrombocytopenia) and requirement for treatment. Seven patients received single-agent therapy, with amelioration of symptoms; 4 patients were observed. There were no disease-associated deaths or progression to more aggressive disease during the follow-up interval (median, 17 months). CONCLUSIONS: Patients with CLPD-NK have an indolent clinical course and frequent hematologic manifestations that are responsive to single-agent therapy. Mutations in STAT3 are common and portend more pronounced clinical manifestations.
Assuntos
Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Transtornos Linfoproliferativos/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Biópsia , Medula Óssea/patologia , Doença Crônica , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Janus Quinases/genética , Leucemia Linfocítica Granular Grande/diagnóstico , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/metabolismo , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we prospectively screened 38 patients with inclusion body myositis for the presence of expanded large granular lymphocyte populations by standard clinical laboratory methods (flow cytometry, examination of blood smears, and T cell receptor gene rearrangements), and performed muscle immunohistochemistry for CD8, CD57, and TIA1. Most (22/38; 58%) patients with inclusion body myositis had aberrant populations of large granular lymphocytes in their blood meeting standard diagnostic criteria for T cell large granular lymphocytic leukaemia. These T cell populations were clonal in 20/20 patients and stably present on follow-up testing in 15 patients a median of 350 days later. T cell aberrant loss of CD5 or gain of expression of CD16 and CD94 were common (19/42, 45%). In comparison, 2/15 (14%) age-matched patients with dermatomyositis, polymyositis, or necrotizing myopathy, and 0/20 (0%) age-matched healthy subjects had large granular lymphocyte expansions, with none of these patients having T cell aberrant expression of CD5, CD16 or CD94. Reduced blood CD4/CD8 ratio, increased blood CD8 count, and lymphocytosis were additional biomarkers highly correlated with flow cytometry-measured large granular lymphocyte expansions. Cross-sectional data suggested more aggressive disease in patients with such expansions than without. Muscle immunohistochemistry demonstrated invasion of large granular lymphocytes into muscle in 15/15 inclusion body myositis patients but in only 1/28 patients with dermatomyositis or polymyositis. The extent of CD8+ and CD57+ cells in inclusion body myositis muscle correlated with the size of blood large granular lymphocyte populations. Myofibre-invading cells expressed CD57, a marker of persistent T cell exposure to antigen and T cell aggressiveness. In many patients with inclusion body myositis, the autoimmune T cell expansion has evolved into a neoplastic-like or overtly neoplastic disorder, perhaps contributing to its relative refractoriness to immune-directed therapies previously reported.
Assuntos
Antígenos CD/metabolismo , Leucemia Linfocítica Granular Grande/complicações , Leucemia Linfocítica Granular Grande/metabolismo , Miosite de Corpos de Inclusão/complicações , Miosite de Corpos de Inclusão/metabolismo , Adulto , Idoso , Antígenos CD/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Humanos , Linfocitose/complicações , Pessoa de Meia-Idade , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculos/citologia , Músculos/metabolismo , Estudos Prospectivos , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
The pathogenesis of T-cell large granular lymphocytic leukemia (T-LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non-synonymous alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T-LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T-LGL that affect about 8% of cases, likely contributing to deregulated NF-κB activity in this leukemia.
Assuntos
Proteínas de Ligação a DNA/genética , Variação Genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Linfocítica Granular Grande/genética , Proteínas Nucleares/genética , Estudos de Coortes , Variações do Número de Cópias de DNA , Proteínas de Ligação a DNA/metabolismo , Éxons , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Mutação , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Fator de Transcrição STAT3/genética , Análise de Sequência de DNA , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Natural killer (NK) large granular lymphocyte (LGL) leukaemia features a clonal proliferation of CD3(-) NK cells that can be classified into either aggressive or chronic categories. The NKL cell line, derived from an aggressive Asian NK cell leukaemia, and patient samples from chronic NK-LGL leukaemia were used in our study to probe for synergistic efficacy of the epigenetic drugs vorinostat (SAHA) and cladribine in this disease. We demonstrate that histone deacetylases (HDACs) are over-expressed in both aggressive and chronic NK leukaemia. Administration of the HDAC inhibitor SAHA reduces class I and II HDAC expression and enhances histone acetylation in leukaemic NK cells. In vitro combination treatment with SAHA and cladribine dose-dependently exerts synergistic cytotoxic and apoptotic effects on leukaemic NK cells. Expression profiling of apoptotic regulatory genes suggests that both compounds led to caspase-dependent apoptosis through activation of intrinsic mitochondrial and extrinsic death receptor pathways. Collectively, these data show that combined epigenetic therapy, using HDAC and DNA methyltransferase inhibitors, may be a promising therapeutic approach for NK-LGL leukaemia.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Leucemia Linfocítica Granular Grande/tratamento farmacológico , Acetilação/efeitos dos fármacos , Apoptose/genética , Cladribina/administração & dosagem , Cladribina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacologia , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais Cultivadas/efeitos dos fármacos , VorinostatRESUMO
The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.
Assuntos
Janus Quinases/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Idoso , Células Cultivadas , Feminino , Humanos , Janus Quinases/genética , Leucemia Linfocítica Granular Grande/genética , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Fosforilação , Fatores de Transcrição STAT/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
The etiology of chronic large granular lymphocyte proliferations is largely unknown. Although these disorders are characterized by the expansion of different cell types (T and natural killer) with specific genetic features and abnormalities, several lines of evidence suggest a common pathogenetic mechanism. According to this interpretation, we speculated that in patients with natural killer-type chronic lymphoproliferative disorder, together with natural killer cells, also T lymphocytes undergo a persistent antigenic pressure, possibly resulting in an ultimate clonal T-cell selection. To strengthen this hypothesis, we evaluated whether clonal T-cell populations were detectable in 48 patients with killer immunoglobulin-like receptor-restricted natural killer-type chronic lymphoproliferative disorder. At diagnosis, in half of the patients studied, we found a clearly defined clonal T-cell population, despite the fact that all cases presented with a well-characterized natural killer disorder. Follow-up analysis confirmed that the TCR gamma rearrangements were stable over the time period evaluated; furthermore, in 7 patients we demonstrated the appearance of a clonal T subset that progressively matures, leading to a switch between killer immunoglobulin-like receptor-restricted natural killer-type disorder to a monoclonal T-cell large granular lymphocytic leukemia. Our results support the hypothesis that a common mechanism is involved in the pathogenesis of these disorders.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia Linfocítica Granular Grande/imunologia , Receptores KIR/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Estudos de Coortes , Progressão da Doença , Feminino , Citometria de Fluxo , Seguimentos , Rearranjo Gênico , Humanos , Imunofenotipagem , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores KIR/genética , Linfócitos T/metabolismo , Linfócitos T/patologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologiaRESUMO
T-cell large granular lymphocytic leukemia (T-LGLL) is a rare indolent lymphoproliferative disorder characterized by cytopenias, splenomegaly, and various degrees of T-cell lymphocytosis, due to a clonal expansion of CD8-positive cytotoxic T-cells. Phenotypic variants of T-LGLL include CD4(+) /CD8(-) T-cells, with dual CD4(-) /CD8(-) /γδ(+) T-cells being even rarer. Cytogenetic abnormalities in T-LGLL have rarely been reported, and there is scientific debate regarding the existence of aggressive or transformed variants of T-LGLL. We report a patient with T-LGLL, γδ variant, with nearly 20-year-long duration of cytopenias before transformation to an unusual clinical scenario, manifesting with marked lymphocytosis >100 × 10(9) /L and infiltration of lymph nodes, tonsils, and subcutaneous tissue. Single-nucleotide polymorphism assays revealed acquired copy neutral loss of heterozygosity at 17q and deletion of 3p21.31, in addition to trisomy 5, monosomy X, and monosomy 21. These genetic abnormalities provided a better understanding of the molecular nature and the potentiality of disease transformation.
Assuntos
Aberrações Cromossômicas , Leucemia Linfocítica Granular Grande/diagnóstico , Leucemia Linfocítica Granular Grande/patologia , Linfócitos T/patologia , Idoso , Antígenos CD/genética , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 5 , Síndrome de Cri-du-Chat , Diagnóstico Diferencial , Feminino , Expressão Gênica , Humanos , Cariotipagem , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Perda de Heterozigosidade , Monossomia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/metabolismo , Trissomia , Síndrome de TurnerAssuntos
Leucemia Linfocítica Granular Grande/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Neoplasias Cutâneas/metabolismo , Linfócitos T/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Feminino , Humanos , Leucemia Linfocítica Granular Grande/complicações , Leucemia Linfocítica Granular Grande/patologia , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/genética , Remissão Espontânea , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologiaAssuntos
Leucemia Linfocítica Granular Grande/genética , Mutação , Proteínas de Neoplasias/genética , Fator de Transcrição STAT3/genética , Animais , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/metabolismo , Fator de Transcrição STAT3/metabolismoAssuntos
Linfócitos T CD4-Positivos , Leucemia Linfocítica Granular Grande/genética , Mutação , Proteínas de Neoplasias/genética , Fator de Transcrição STAT5/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Incidência , Leucemia Linfocítica Granular Grande/epidemiologia , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
Oligo-array comparative genomic hybridization (CGH) and gene-expression profiling of natural killer (NK)-cell neoplasms were used in an effort to delineate the molecular pathogenesis involved. Oligo-array CGH identified two 6q21 regions that were most frequently deleted (14 of 39 or 36%). One of these regions included POPDC3, PREP, PRDM1, ATG5, and AIM1, whereas the other included LACE1 and FOXO3. All genes located in these regions, except for POPDC3 and AIM1, were down-regulated in neoplastic samples, as determined by gene-expression analysis, and were therefore considered to be candidate tumor-suppressor genes. A20 and HACE1, the well-known tumor-suppressor genes located on 6q21-23, were included as candidate genes because they also demonstrated frequent genomic deletions and down-regulated expression. The Tet-Off NK cell line NKL was subsequently established for functional analyses. Seven candidate genes were transduced into Tet-Off NKL and forced re-expression was induced. Re-expression of FOXO3 and PRDM1 suppressed NKL proliferation, but this was not the case after re-expression of the other genes. This effect was confirmed using another NK cell line, SNK10. Furthermore, genomic analyses detected nonsense mutations of PRDM1 that led to functional inactivation in one cell line and one clinical sample. PRDM1 and FOXO3 are considered to play an important role in the pathogenesis of NK-cell neoplasms.
Assuntos
Hibridização Genômica Comparativa/métodos , Fatores de Transcrição Forkhead , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genômica/métodos , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Granular Grande/genética , Linfoma Folicular/genética , Adolescente , Adulto , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Criança , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genoma Humano , Humanos , Células Matadoras Naturais/patologia , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Retroviridae , Transdução GenéticaAssuntos
Leucemia Linfocítica Granular Grande , Doenças do Sistema Nervoso Periférico , Humanos , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Leucemia Linfocítica Granular Grande/fisiopatologia , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/fisiopatologiaRESUMO
The blood cancer T cell large granular lymphocyte (T-LGL) leukemia is a chronic disease characterized by a clonal proliferation of cytotoxic T cells. As no curative therapy is yet known for this disease, identification of potential therapeutic targets is of immense importance. In this paper, we perform a comprehensive dynamical and structural analysis of a network model of this disease. By employing a network reduction technique, we identify the stationary states (fixed points) of the system, representing normal and diseased (T-LGL) behavior, and analyze their precursor states (basins of attraction) using an asynchronous Boolean dynamic framework. This analysis identifies the T-LGL states of 54 components of the network, out of which 36 (67%) are corroborated by previous experimental evidence and the rest are novel predictions. We further test and validate one of these newly identified states experimentally. Specifically, we verify the prediction that the node SMAD is over-active in leukemic T-LGL by demonstrating the predominant phosphorylation of the SMAD family members Smad2 and Smad3. Our systematic perturbation analysis using dynamical and structural methods leads to the identification of 19 potential therapeutic targets, 68% of which are corroborated by experimental evidence. The novel therapeutic targets provide valuable guidance for wet-bench experiments. In addition, we successfully identify two new candidates for engineering long-lived T cells necessary for the delivery of virus and cancer vaccines. Overall, this study provides a bird's-eye-view of the avenues available for identification of therapeutic targets for similar diseases through perturbation of the underlying signal transduction network.
Assuntos
Leucemia Linfocítica Granular Grande/imunologia , Leucemia Linfocítica Granular Grande/metabolismo , Linfócitos T Citotóxicos/metabolismo , Sobrevivência Celular/imunologia , Humanos , Fosforilação , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferative disorder of cytotoxic lymphocytes characterized by an expansion of CD3(+) cytotoxic T lymphocytes or CD3(-) natural killer cells. Patients present with various cytopenias including neutropenia, anemia and thrombocytopenia. In addition, there is an association of T-cell large granular lymphocytic leukemia with rheumatoid arthritis. It is believed that LGL leukemia begins as an antigen-driven immune response with subsequent constitutive activation of cytotoxic T lymphocytes or natural killer cells through PDGF and IL-15 contributing to their survival. Consequently, this leads to a dysregulation of apoptosis and dysfunction of the activation-induced cell death pathway. Treatment of LGL leukemia is based on a low-dose immunosuppressive regimen using methotrexate or cyclophosphamide. However, no standard of therapy has been established, as large prospective trials have not been conducted. In addition, some patients are refractory to treatment. The lack of a curative therapy for LGL leukemia means that new treatment options are needed. Insight into the various dysregulated signaling pathways in LGL leukemia may provide novel therapeutic treatment modalities.
Assuntos
Ciclofosfamida/uso terapêutico , Imunossupressores/uso terapêutico , Leucemia Linfocítica Granular Grande/tratamento farmacológico , Leucemia Linfocítica Granular Grande/imunologia , Leucemia Linfocítica Granular Grande/metabolismo , Metotrexato/uso terapêutico , Apoptose/imunologia , Proteína Ligante Fas/metabolismo , Humanos , Imunossupressores/administração & dosagem , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteínas ras/metabolismoRESUMO
Cytokine-dependent cell lines have been used to analyze the cytokine-induced cellular signaling and the mechanism of oncogenesis. In the current study, we analyzed MOTN-1 and PLT-2 cell lines established from different stages of a T-cell large granular lymphocyte leukemia patient (Daibata et al. 2004). MOTN-1 is IL-2-dependent derived from the chronic phase, whereas IL-2-independent PLT-2 is from the aggressive and terminal stage. They shared considerable chromosome abnormalities and the pattern of T-cell receptor rearrangement, presuming that the cytokine independence of PLT-2 was due to the additive genetic abnormality. Besides IL-2, IL-15 supported MOTN-1 cell growth, because these receptors share beta- and gamma-subunits. IL-2 activated ERK, AKT and STAT pathway of MOTN-1. STAT3 pathway of PLT-2 was also activated by IL-2, suggesting intact IL-2 induces signal transduction of PLT-2. However, ERK1/2 but not AKT, was continuously activated in PLT-2, consistent with the increased Ras-activity of PLT-2. Sequence analysis revealed KRAS G12A mutation but not NRAS and HRAS mutation of PLT-2 but not MOTN-1. Another signaling molecule affecting Ras-signaling pathway, SHP2, which has been frequently mutated in juvenile myelomonocytic leukemia (JMML), did not show mutation. Moreover, MEK inhibitor, PD98059, as well as farnesylation inhibitor inhibited PLT-2 cell growth. Using NIH3T3 and MOTN-1, ERK activation, increased cell proliferation and survival by KRAS G12A were shown, suggesting the important role of KRAS G12A in IL-2-independent growth of PLT-2. Taken together, KRAS G12A is important for IL-2-independent growth of PLT-2 cells and suggests the possibility of involvement of KRAS mutation with disease progression.
Assuntos
Interleucina-2/farmacologia , Leucemia Linfocítica Granular Grande/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Flavonoides/farmacologia , Humanos , Interleucina-15/farmacologia , Leucemia Linfocítica Granular Grande/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
OBJECTIVE: To explore the hematopathologic features of T-cell large granular lymphocytic leukemia (T-LGLL). METHODS: A retrospective analysis of the clinical presentation, bone marrow morphology, immunophenotyping and T-cell receptor gene rearrangement status were performed in 19 patients with T-LGLL. RESULTS: Of 19 patients, the most frequent hematological abnormalities were anemia and neutropenia (16/19 and 17/19 patients, respectively). Large granular lymphocytes (LGLs) were observed in 17 of 19 peripheral blood smears and 15 of 19 bone marrow aspirate specimens. Lymphocytosis (> 0.2) was present in 17 of 19 patients in their bone marrow aspirate specimens. Bone marrow biopsy specimens revealed lymphocytosis in 16 cases, with a mild to moderate increase of lymphocytes observed in 12 cases (12/16). The pattern of lymphoid distribution was interstitial in bone marrow sections. Intravascular distribution was seen in 8 cases. Lymphoid nodules were present in 4 cases. Flow cytometery showed an immunophenotype of CD3(+) CD4(-) CD8(+) CD56(-) CD57(+) of the tumor cells in 13 cases. Of the other 6 cases, the immunophenotypes included CD8(-) (1 case), CD56(+) (2 cases) and CD57(-) (3 cases). Immunohistochemistry showed CD3+ (10/10), CD57+ (3/3), CD8+ (6/7), TIA-1+ (6/7), granzyme B+ (4/7), perforin + (1/7), CD4- (4/4) and CD56- (9/9). Clonal T-cell receptor γ gene rearrangement by PCR was detected in 12 cases (12/17). CONCLUSIONS: Hematopathologic features of most T-LGLL are distinct. Morphologic, immunophenotypic and molecular analysis of both peripheral blood and bone marrow specimens are essential and complementary in the diagnosis and differential diagnosis of T-LGLL.
Assuntos
Anemia/patologia , Medula Óssea/patologia , Leucemia Linfocítica Granular Grande/patologia , Linfocitose/patologia , Neutropenia/patologia , Adulto , Idoso , Anemia/metabolismo , Complexo CD3/metabolismo , Antígenos CD57/metabolismo , Antígenos CD8/metabolismo , Diagnóstico Diferencial , Feminino , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Granzimas/metabolismo , Humanos , Imunofenotipagem , Leucemia Linfocítica Granular Grande/metabolismo , Linfocitose/metabolismo , Masculino , Pessoa de Meia-Idade , Neutropenia/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Estudos Retrospectivos , Antígeno-1 Intracelular de Células TRESUMO
BACKGROUND: The molecular pathogenesis of T-cell large granular lymphocytic leukemia (T-LGLL), a mature T-cell leukemia arising commonly from T-cell receptor αß-positive CD8+ memory cytotoxic T cells, is only partly understood. The role of deregulated methylation in T-LGLL is not well known. We analyzed the epigenetic profile of T-LGLL cells of 11 patients compared to their normal counterparts by array-based DNA methylation profiling. For identification of molecular events driving the pathogenesis of T-LGLL, we compared the differentially methylated loci between the T-LGLL cases and normal T cells with chromatin segmentation data of benign T cells from the BLUEPRINT project. Moreover, we analyzed gene expression data of T-LGLL and benign T cells and validated the results by pyrosequencing in an extended cohort of 17 patients, including five patients with sequential samples. RESULTS: We identified dysregulation of DNA methylation associated with altered gene expression in T-LGLL. Since T-LGLL is a rare disease, the samples size is low. But as confirmed for each sample, hypermethylation of T-LGLL cells at various CpG sites located at enhancer regions is a hallmark of this disease. The interaction of BLC11B and C14orf64 as suggested by in silico data analysis could provide a novel pathogenetic mechanism that needs further experimental investigation. CONCLUSIONS: DNA methylation is altered in T-LGLL cells compared to benign T cells. In particular, BCL11B is highly significant differentially methylated in T-LGLL cells. Although our results have to be validated in a larger patient cohort, BCL11B could be considered as a potential biomarker for this leukemia. In addition, altered gene expression and hypermethylation of enhancer regions could serve as potential mechanisms for treatment of this disease. Gene interactions of dysregulated genes, like BLC11B and C14orf64, may play an important role in pathogenic mechanisms and should be further analyzed.