RESUMO
Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.
Assuntos
Linfócitos B/patologia , Biomarcadores Tumorais/genética , Genômica/métodos , Leucemia Linfocítica Crônica de Células B/genética , Linfocitose/genética , Mutação , Estudos de Coortes , Progressão da Doença , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfocitose/patologia , Fenótipo , Prognóstico , Sequenciamento Completo do GenomaRESUMO
Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)-like, atypical CLL, and CD5(-) MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5(-) MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor ß (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4(high)CD8(low), CD8(high)CD4(low) T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.
Assuntos
Linfócitos B/metabolismo , Aberrações Cromossômicas , Leucemia Linfocítica Crônica de Células B/genética , Linfocitose/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/patologia , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 17/genética , Progressão da Doença , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Contagem de Linfócitos , Linfocitose/diagnóstico , Linfocitose/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Recombinação V(D)J/genéticaRESUMO
We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27, a key regulator of cell cycle, in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly, expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly, when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients, increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition, when CLL cells were induced in vitro to enter cell cycle (eg, with cytosine phosphate guanine oligodeoxynucleotide), a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition.
Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Western Blotting , Ciclo Celular , Imunofluorescência , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2(-/-)gamma(c)(-/-) mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.
Assuntos
Proteínas de Ligação a DNA , Modelos Animais de Doenças , Leucemia Linfocítica Crônica de Células B , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transplante de Neoplasias , Transplante HeterólogoRESUMO
The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eµ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eµ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/patologia , Proteínas Sanguíneas/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/citologia , Proteínas Sanguíneas/genética , Medula Óssea/patologia , Adesão Celular , Linhagem Celular Tumoral , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
CLL-like monoclonal B-cell lymphocytosis (MBL) shares a unique immunophenotype with chronic lymphocytic leukemia (CLL), and represents the vast majority of clonal B-cell expansions found in the peripheral blood of otherwise healthy subjects. Along with the improvement of laboratory techniques and the widespread availability of multiparameter flow cytometry, the finding of tiny aberrant B-cell populations became more frequent, prompting the need for clinical and biological definition of the nature of this condition and its relationship with leukemia development. MBL seems to be a melting-pot containing several entities, identical in terms of phenotype but with extremely different risks of leukemia development (from low to none) that seem to correlate with the number of B lymphocytes. CLL-like MBL observed in the clinical setting ("Clinical MBL"), usually being characterized by lymphocytosis, demonstrated a sizeable, even if low (1.1-1.4% per year), risk of leukemic progression, but represents a minority of all MBL cases. The vast majority of CLL-like MBL are detected in general population screenings and do not likely have a risk of CLL that is substantially higher than that of unaffected individuals. Interestingly, MBL frequency increases with age, being virtually undetectable under 40 years of age but being present in 50-75% of the people older than 90 years. It has been proposed that MBL could be interpreted as an epiphenomenon of a chronic and persistent antigenic stimulation. The (rare) possibility to evolve into a frank leukemia might then depend on biological and molecular factors insofar unknown that may modify the modality of cell reaction as well as the potential to acquire further genetic abnormalities. Therefore, the real challenge of the next years in the MBL research field is not to increase the sensitivity of detection, neither to implement screening protocols to be applied to the general population, rather to unravel the biologic features that, at individual level, will identify those (few) cases that are at risk of developing a progressive disease.
Assuntos
Linfócitos B/imunologia , Linfocitose/imunologia , Distribuição por Idade , Animais , Linfócitos B/patologia , Senescência Celular , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfocitose/patologiaRESUMO
In the revised National Cancer Institute Working Group (NCI-WG)/International Workshop on Chronic Lymphocytic Leukemia (IWCLL) guidelines for CLL, CLL-like monoclonal B lymphocytosis (MBL) is defined as the presence of less than 5 x 10(9)/L B lymphocytes in the peripheral blood. However, the concentration of MBL in the blood is extremely variable. MBL in subjects with lymphocytosis require treatment at a rate of 1.1% per year and present immunoglobulin (IG) gene features and similar to good prognosis CLL. Little is known about low-count MBL cases, accidentally found in the general population. We analyzed IGHV-D-J rearrangements in 51 CLL-like MBL cases from healthy individuals, characterized by few clonal B cells. Seventy percent of the IGHV genes were mutated. The most frequent IGHV gene was IGHV4-59/61, rarely used in CLL, whereas the IGHV1-69 gene was lacking and the IGHV4-34 gene was infrequent. Only 2 of 51 (3.9%) MBL cases expressed a CLL-specific stereotyped HCDR3. Therefore, the IG gene repertoire in low-count MBL differs from both mutated and unmutated CLL, suggesting that the detection of MBL in an otherwise healthy subject is not always equivalent to a preleukemic state. Detailed IG analysis of individual MBL may help to identify cases that necessitate continuous clinical monitoring to anticipate disease progression.
Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Linfocitose/genética , Linfocitose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Regiões Determinantes de Complementaridade , Diagnóstico Diferencial , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Itália , Leucemia Linfocítica Crônica de Células B/diagnóstico , Contagem de Linfócitos , Linfocitose/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Pré-Leucemia/diagnóstico , Pré-Leucemia/genética , Pré-Leucemia/imunologia , Adulto JovemAssuntos
Leucemia Linfocítica Crônica de Células B/epidemiologia , Linfoma Folicular/epidemiologia , Gamopatia Monoclonal de Significância Indeterminada/epidemiologia , Mieloma Múltiplo/epidemiologia , Lesões Pré-Cancerosas/epidemiologia , Adulto , Idoso , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/ultraestrutura , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 18/ultraestrutura , Progressão da Doença , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Pessoa de Meia-Idade , Prevalência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Taiwan/epidemiologia , Translocação Genética , Adulto JovemRESUMO
PURPOSE: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. EXPERIMENTAL DESIGN: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL. RESULTS: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02-4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05-4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08-5.2; P = 0.03). CONCLUSIONS: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.
Assuntos
Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/genética , Mutação , Neoplasias Esplênicas/diagnóstico , Neoplasias Esplênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Feminino , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição Kruppel-Like/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Prognóstico , Receptor Notch2/genética , Fatores de Risco , Fatores de Transcrição/genética , Resultado do Tratamento , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Proteína Supressora de Tumor p53/genéticaRESUMO
Monoclonal B-cell lymphocytosis (MBL) is defined as a clonal B-cell expansion whereby the B-cell count is less than 5 × 10(9)/L and no symptoms or signs of lymphoproliferative disorders are detected. Based on B-cell count, MBL is further divided into low-count and clinical MBL. While low-count MBL seems to carry relevance mostly from an immunological perspective, clinical MBL and chronic lymphocytic leukemia appear to be overlapping entities. Only a deeper knowledge of molecular pathways and microenvironmental influences involved in disease evolution will help to solve the main clinical issue, i.e. how to differentiate nonprogressive and progressive cases requiring intensive follow-up.
Assuntos
Linfócitos B/metabolismo , Leucemia Linfocítica Crônica de Células B/diagnóstico , Linfocitose/diagnóstico , Animais , Linfócitos B/imunologia , Senescência Celular/imunologia , Diagnóstico Diferencial , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Contagem de Linfócitos , Linfocitose/imunologia , Estadiamento de NeoplasiasRESUMO
PURPOSE: The emerging role of Toll-like receptors (TLR) in the pathogenesis of chronic lymphocytic leukemia (CLL) led us to ask whether TLR stimulation may protect CLL cells from drug-induced apoptosis. EXPERIMENTAL DESIGN: We cultured in vitro malignant B cells freshly isolated from 44 patients with CLLs in the presence or the absence of different concentrations of fludarabine before or after 24-hour TLR stimulation with specific ligands and evaluated cell viability, apoptosis, and molecular pathways involved. RESULTS: Heterogeneity was observed among samples. In leukemic cells from patients bearing adverse prognostic factors, TLR stimulation caused a significant increase of protection to fludarabine treatment, whereas this did not occur in the cells from patients with good prognosis. To identify novel molecular mechanisms accounting for the dichotomy of response between the two groups of patients, we conducted an apoptosis gene expression profile on leukemic cells either unstimulated or stimulated with TLR9 ligand. Strikingly, TLR9 stimulation specifically upregulated the expression of lymphotoxin-α in cells where an increased protection to fludarabine treatment was observed. Also, the expression of miR-155-3p was significantly increased after stimulation of distinct TLR in cells where fludarabine treatment was less effective. CONCLUSIONS: These results suggest that at least in a proportion of patients, in vitro sensitivity to fludarabine may be modulated by the stimulation of TLR, likely mimicking microenvironmental signals occurring in vivo.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptores Toll-Like/metabolismo , Vidarabina/análogos & derivados , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Linfotoxina-alfa/genética , Linfotoxina-alfa/metabolismo , MicroRNAs/genética , Prognóstico , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Vidarabina/farmacologiaRESUMO
BACKGROUND: Monoclonal B cell lymphocytosis (MBL) is a preclinical condition characterized by an expansion of clonal B cells in the absence of B lymphocytosis (BALC < 5 × 10(9)/L) in the peripheral blood, without clinical signs, suggestive of a lymphoproliferative disorder. B cell clonal expansions are also associated with hepatitis C virus (HCV) infection and they can evolve into lymphoproliferative disorders such as mixed cryoglobulinemia and non-Hodgkin lymphomas (NHL). The relationship between MBL and HCV infection has not been established yet. METHODS: By five-colour flow cytometry, we analyzed 123 HCV positive subjects with diagnosis of chronic hepatitis (94) or cirrhosis (29); 16 of those with cirrhosis had a diagnosis of hepatocellular carcinoma. RESULTS: MBL were identified in 35/123 (28.5%), at significantly higher frequency than in the general population. Sixteen/thirty-five were atypical-chronic lymphocytic leukemia (CLL) MBL (CD5(+), CD20(bright)), 13/35 were CLL-like MBL (CD5(bright), CD20(dim)), and 6/35 were CD5(-) MBL. Twenty-four/ninety-four (25.5%) patients affected by chronic hepatitis had MBL, whereas 11/29 (37.9%) patients with cirrhosis showed a B cell clone. A biased usage of IGHVgenes similar to HCV-associated NHL was evident. CONCLUSIONS: All three types of MBL can be identified in HCV-infected individuals at a higher frequency than in the general population, and their presence appears to correlate with a more advanced disease stage. The phenotypic heterogeneity is reminiscent of the diversity of NHL arising in the context of HCV infection. The persistence of HCV may be responsible for the dysregulation of the immune system and in particular of the B cell compartment.
Assuntos
Linfócitos B/patologia , Hepatite C Crônica/patologia , Leucocitose/patologia , Cirrose Hepática/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Linfócitos B/virologia , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Células Clonais , Feminino , Citometria de Fluxo , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Hepacivirus/genética , Hepacivirus/imunologia , Hepacivirus/isolamento & purificação , Hepatite C Crônica/etiologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem , Leucocitose/etiologia , Cirrose Hepática/etiologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Monoclonal B lymphocytosis (MBL) is a frequent phenomenon in the general population. Despite a phenotype similar to chronic lymphocytic leukemia (CLL), the possibility exists that most cases are not necessarily a pre-leukemic condition. This is suggested by the fact that MBL is at least 100 times more frequent than CLL and the diagnosis of CLL is not an inevitable fate, even among MBL cases with lymphocytosis, where it occurs only in 1.1% of the cases per year. The high incidence of MBL, if coupled with the possibility of evolution into a frank leukemic state, poses evident clinical and health system concerns. MBL in the general population usually accounts for a very low number of all circulating B-cells, being <10% of all B lymphocytes. This creates the need for a better characterisation of MBL at molecular level, aiming to identify biological features that may define which cases are more likely to progress towards clinically overt CLL. This approach should also help to avoid unnecessary and prolonged follow-ups in all individuals carrying MBL, excluding those who are extremely unlikely to develop CLL.
Assuntos
Linfócitos B/patologia , Linfocitose/epidemiologia , Células Clonais , Humanos , Incidência , Itália/epidemiologia , Linfocitose/diagnóstico , Programas de RastreamentoRESUMO
Hemojuvelin (HJV) positively modulates the iron regulator hepcidin, and its mutations are the major cause of juvenile hemochromatosis (JH), a recessive disease leading to iron overload. Defective HJV reduces hepcidin up-regulation both in humans and in Hjv-deficient mice. To investigate the JH pathogenesis and the functional properties of human HJV we studied the biosynthesis and maturation of 6 HJV pathogenic mutants in HeLa and HepG2 cells. We show that proteolytic processing is defective in mutants F170S, W191C, and G320V, but not in G99V and C119F. Moreover, we show that mutants G99V and C119F are targeted to the cell surface, while F170S, W191C, G320V, and R326X (lacking the glycosilphosphatidylinositol [GPI] anchor) are mainly retained in the endoplasmic reticulum, although all mutants are released as soluble forms (s-HJV) in a proportion that is modulated by iron supplementation. Membrane HJV (m-HJV) is mainly composed of the cleaved protein, and its level is increased by iron in wild-type (WT) mice but not in the mutants. Altogether, the data demonstrate that the loss of HJV membrane export is central to the pathogenesis of JH, and that HJV cleavage is essential for the export. The results support a dual function for s- and m-HJV in iron deficiency and overload, respectively.