RESUMO
Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.
Assuntos
Biomarcadores Tumorais/genética , Linfoma Folicular/genética , Mutação , Complexo Repressor Polycomb 2/genética , Proteína de Ligação a CREB/genética , Estudos de Coortes , Análise Mutacional de DNA , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Perfilação da Expressão Gênica , Frequência do Gene , Humanos , Estimativa de Kaplan-Meier , Linfoma Folicular/patologia , Fatores de Transcrição MEF2/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Fatores de TempoRESUMO
Despite the use of highly active antiretroviral therapy (HAART), AIDS-related lymphoma remains common. We investigated the tumor, microenvironment, and viral components in 41 AIDS-related diffuse large B-cell lymphomas (AR-DLBCLs) in the pre- and post-HAART era. The outcome has improved and the frequency of the prognostically unfavorable immunoblastic histology has decreased after HAART. Compared with sporadic cases, AR-DLBCL demonstrated increased hyperproliferation (P < .001) and c-Myc rearrangements, reduced CD4(+) (P < .001) and FOXP3(+) T cells (P < .001), increased activated cytotoxic cells (P < .001), but no difference in tumor-associated macrophages. Our analysis showed that AR-DLBCL is highly angiogenic with higher blood-vessel density than sporadic cases (P < .001) and highlighted the role of Epstein-Barr virus in angiogenesis. We recognized viral profiles and as a second step examined the reactive cytotoxic cell infiltrates. Our observation of markedly higher numbers of cytotoxic cells in AR-DLBCL with LMP1 and/or p24 compared with cases lacking viral antigens (P < .001) has important clinical implications, implicitly linked to the immunosurveillance theory. Whereas early initiation of HAART should improve immunosurveillance and reduce the incidence of LMP1-positive AR-DLBCL, cases without viral antigens appear able to avoid immunologic reaction and likely require additional strategies to improve surveillance.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/patologia , Linfoma Relacionado a AIDS/tratamento farmacológico , Linfoma Relacionado a AIDS/patologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Microambiente Tumoral , Síndrome da Imunodeficiência Adquirida/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Vasos Sanguíneos/patologia , Senescência Celular/imunologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/patologia , Feminino , Rearranjo Gênico/genética , HIV-1/fisiologia , Herpesvirus Humano 4/fisiologia , Humanos , Linfócitos do Interstício Tumoral/patologia , Linfócitos do Interstício Tumoral/virologia , Linfoma Difuso de Grandes Células B/complicações , Linfoma Difuso de Grandes Células B/virologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/complicações , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adulto JovemRESUMO
AIMS: To investigate the relationship between Bcl-2 protein expression and cell proliferation at single-cell level in B-cell lymphomas using double-labelling techniques. METHODS AND RESULTS: The relationship between Bcl-2 protein expression and cell proliferation was explored in 124 cases of B-cell lymphoma using double immunofluorescence labelling for Bcl-2 and Ki67. In follicular lymphoma, marginal zone lymphoma and a subset of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), neoplastic cells tended to lose Bcl-2 when they are in cell cycle. This pattern is usually maintained in both follicular lymphoma and CLL/SLL when they undergo high-grade transformation. In mantle cell lymphoma, diffuse large B-cell lymphoma and a subset of CLL/SLL, the inverse relationship (between Bcl-2 and Ki67) was not observed, i.e. the proliferating cells tended to show co-expression of Bcl-2. CONCLUSIONS: In low-grade lymphomas, including those that are transformed, Bcl-2 expression is lost when cell proliferate. However, in more aggressive tumours (i.e. mantle cell and de novo diffuse large B-cell lymphomas) the inverse Bcl-2/Ki67 relationship was not observed. It would be of interest to explore the clinical implications in lymphoma of the presence and absence of the inverse Bcl-2/Ki67 pattern.
Assuntos
Biomarcadores Tumorais/metabolismo , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proliferação de Células , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Antígeno Ki-67/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/patologia , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologiaRESUMO
Follicular lymphoma has considerable clinical heterogeneity, and there is a need for easily quantifiable prognostic biomarkers. Microvessel density has been shown to be a useful prognostic factor based on numerical assessment of vessel numbers within histologic sections in some studies, but assessment of tumor neovascularization through angiogenic sprouting may be more relevant. We therefore examined the smallest vessels, single-staining structures measuring less than 30 microm(2) in area, seen within histologic sections, and confirmed that they were neovascular angiogenic sprouts using extended focal imaging. Tissue microarrays composing diagnostic biopsies from patients at the extremes of survival of follicular lymphoma were analyzed with respect to numbers of these sprouts. This analysis revealed higher angiogenic activity in the poor prognostic group and demonstrated an association between increased sprouting and elevated numbers of infiltrating CD163(+) macrophages within the immediate microenvironment surrounding the neovascular sprout.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores Tumorais/metabolismo , Linfoma Folicular/patologia , Macrófagos/patologia , Neovascularização Patológica/patologia , Receptores de Superfície Celular/metabolismo , Biópsia , Humanos , Macrófagos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , PrognósticoRESUMO
An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.
Assuntos
Antineoplásicos/farmacologia , Comunicação Celular/imunologia , Sinapses Imunológicas/imunologia , Linfoma Folicular/imunologia , Linfócitos T/imunologia , Talidomida/análogos & derivados , Actinas/imunologia , Comunicação Celular/efeitos dos fármacos , Técnicas de Cocultura , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Sinapses Imunológicas/efeitos dos fármacos , Imunoterapia/métodos , Lenalidomida , Microscopia Confocal , Linfócitos T/efeitos dos fármacos , Talidomida/farmacologia , Análise Serial de Tecidos , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab27 de Ligação ao GTPRESUMO
Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.
Assuntos
Crise Blástica/imunologia , Sinapses Imunológicas/imunologia , Leucemia Mieloide Aguda/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/sangue , Crise Blástica/diagnóstico , Crise Blástica/genética , Crise Blástica/patologia , Complexo CD3 , Antígenos CD36 , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/imunologia , Genótipo , Humanos , Sinapses Imunológicas/genética , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/patologia , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)-positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 'pseudo-negative' cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these 'pseudo-negative' cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo-negative immunostaining.
Assuntos
Biomarcadores Tumorais/análise , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Linfoma Folicular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/análise , Translocação Genética , Proliferação de Células , Análise Mutacional de DNA , Genes bcl-2 , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente/métodos , Antígeno Ki-67/análise , Linfoma Folicular/genética , MutaçãoRESUMO
Cancer is associated with immune deficiency, but the biologic basis of this is poorly defined. Here we demonstrate that impaired actin polymerization results in CD4+ and CD8+ T cells from patients with chronic lymphocytic leukemia (CLL) exhibiting defective immunological synapse formation with APCs. Although this synapse dysfunction was in part a result of the CLL cells having poor APC function, defective actin polymerization was also identified in T cells from patients with CLL. We further demonstrate that, following contact with CLL cells, defects in immune synapse formation were induced in healthy allogeneic T cells. This required direct contact and was inhibited by blocking adhesion molecules on CLL B cells. In T cells from patients with CLL and in T cells from healthy individuals that had been in contact with CLL cells, recruitment of key regulatory proteins to the immune synapse was inhibited. Treatment of autologous T cells and CLL cells with the immunomodulating drug lenalidomide resulted in improved synapse formation. These results define what we believe to be a novel immune dysfunction in T cells from patients with CLL that has implications for both autologous and allogeneic immunotherapy approaches and identifies repair of immune synapse defects as an essential step in improving cancer immunotherapy approaches.
Assuntos
Apresentação de Antígeno/imunologia , Fatores Imunológicos/farmacologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos T/imunologia , Actinas/metabolismo , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Humanos , Interleucina-2/metabolismo , Lenalidomida , Leucemia Linfocítica Crônica de Células B/sangue , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Superantígenos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Talidomida/análogos & derivados , Talidomida/farmacologiaRESUMO
PURPOSE: To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance. PATIENTS AND METHODS: Immunohistochemistry was used to study numbers and location of cells staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25). RESULTS: CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location. CONCLUSION: This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.