RESUMO
Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPbeta and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Quinase do Linfoma Anaplásico , Animais , Apoptose/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/fisiopatologia , Camundongos , Antígenos de Histocompatibilidade Menor , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Proteína Tirosina Quinases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.
Assuntos
Movimento Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Regulação para Baixo , Ativação Enzimática , Proteína Adaptadora GRB2/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células K562 , Linfoma Difuso de Grandes Células B/genética , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/genética , RNA Interferente Pequeno/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , TransfecçãoRESUMO
BACKGROUND: We hypothesized that the potent neovascularization marker endoglin (CD105), by differentially highlighting a subset of microvessels (MV) in esophageal cancer (EC), could provide better prognostic/therapeutic information than the panendothelial marker CD34, which also highlights MV. METHODS: Endoglin messenger ribonucleic acid (mRNA) expression in normal, malignant, and adjacent nontumorous esophagus tissue was quantified by real-time reverse-transcription polymerase chain reaction (RT-PCR). Sections of formalin-fixed, paraffin-embedded tissues were analyzed immunohistochemically for CD105 and CD34. MV density was counted following a standard protocol. Circulating soluble endoglin levels were determined in patient and control sera, and compared with clinical outcome. RESULTS: CD105 mRNA was upregulated by a median factor of 2.89 in ECs versus controls. In 28% of patients, CD105 mRNA was upregulated by a median factor of 2.65 in adjacent non-tumorous versus normal tissue. In tumor tissues, CD105 was stained negatively or positively only in a subset of MV. CD34 always showed positive extensive MV staining. In adjacent nontumorous esophagus, CD105 rarely showed diffuse MV staining, while CD34 stained blood-vessel endothelial cells in all non-neoplastic tissue. CD105 expression was high in residual highly dysplastic Barrett's-type mucosa associated with some adenocarcinomas. No statistically significant difference in endoglin serum levels appeared between patients and normal subjects. Correlation with clinicopathological data showed higher intra-tumor MV-CD105+ scores at more-advanced clinical stages. High-scoring MV-CD105+ patients had significantly shorter disease-free and overall survival; MV-CD34+ density was not survival related. Diffuse CD105 expression in adjacent nontumorous esophagus predicted poorer disease-free and overall survival. CONCLUSIONS: Our findings could help identify EC patients who may benefit from targeted anti-angiogenic therapies.
Assuntos
Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Esôfago de Barrett/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Recidiva Local de Neoplasia/diagnóstico , Receptores de Superfície Celular/metabolismo , Adenocarcinoma/genética , Adulto , Idoso , Antígenos CD/genética , Esôfago de Barrett/genética , Carcinoma de Células Escamosas/genética , Diferenciação Celular , Endoglina , Neoplasias Esofágicas/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Masculino , Microcirculação , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.
Assuntos
Citometria de Fluxo/métodos , Linfoma de Células T Periférico/química , Linfoma de Células T Periférico/diagnóstico , Neprilisina/análise , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Diagnóstico Diferencial , Feminino , Humanos , Linfadenopatia Imunoblástica/diagnóstico , Imunofenotipagem , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Pseudolinfoma/diagnóstico , Estudos RetrospectivosRESUMO
The prognostic significance of microvessel density and proliferative activity of the neoplastic cells, evaluated respectively by CD31 and Ki-67 positivity, and immunohistochemical expression of vascular endothelial growth factor (VEGF) was retrospectively investigated in 105 cases of sinonasal carcinoma (80 surgical specimens and 25 biopsies). The most represented histologic types were intestinal-type adenocarcinoma found in 36 patients (34.3%), squamous cell carcinoma (SCC) in 34 (32.4%), mucinous adenocarcinoma (mainly made up of signet-ring cell patterns) in 15 (14.3%), and adenoid cystic carcinoma in 7 (6.7%). Microvessel density values (in vessels per square millimeter), VEGF, and Ki-67 were not dependent on histologic type but were rather correlated to the histologic grading in SCC. Clinical data were available for 92 (87.6%) of 105 patients, with minimum follow-up of 48 months. Most of the patients (81.5%) were at an advanced stage (T3-T4) at diagnosis. The values of all markers were correlated to tumor stage (P = .03). Multivariate analysis showed that both microvessel density and proliferative activity of the neoplastic cells were independent prognostic parameters (mortality hazard ratio, 1.33 and 1.60, respectively). Although VEGF expression was not correlated to prognosis on the whole series (P = .06), it was a powerful prognostic marker when the analysis was restricted to the group of SCCs (hazard ratio, 3.02; 90% confidence interval, 1.58-5.80). These results show that tumor neoangiogenesis, expressed by microvessel density, together with proliferative activity, is a pathologic marker with a strong prognostic impact in sinonasal carcinomas. Therefore, it may be a useful tool in this field so as to carry out therapeutic protocol planning, which may be further enhanced by the adoption of the more recent antiangiogenic molecules.
Assuntos
Adenocarcinoma/irrigação sanguínea , Neovascularização Patológica/patologia , Neoplasias dos Seios Paranasais/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Terapia Combinada , Intervalo Livre de Doença , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Microcirculação/patologia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasias dos Seios Paranasais/metabolismo , Neoplasias dos Seios Paranasais/patologia , Estudos RetrospectivosRESUMO
Microscopic examination of a blood clot expelled by a physically and mentally disabled woman taken to the emergency room because of genital bleeding revealed the presence of chorionic villi encircled by decidua, hemorrhage, and necrosis. In order to identify the father of the product of conception, sections of formalin-fixed, paraffin-embedded abortion material were subjected to laser microdissection: DNA extraction from chorionic villi selectively isolated from the surrounding tissues allowed successful STR-typing of fetal cells, which was otherwise prevented by excess maternal DNA. The large number of homozygous genotypes in the fetal profile suggested incestuous paternity. Analysis of reference DNA samples from male relatives excluded the woman's father, paternal grandfather, and maternal grandfather, whereas the obligate paternal alleles of the fetus were constantly present in the genotypes of the woman's brother, clearly demonstrating brother-sister incest (probability of paternity > 99.99999%).
Assuntos
Feto Abortado/citologia , Vilosidades Coriônicas/patologia , Incesto , Microdissecção/métodos , Paternidade , Sequências de Repetição em Tandem , Adulto , Pessoas com Deficiência , Feminino , Fixadores , Formaldeído , Humanos , Lasers , Masculino , Inclusão em Parafina , Delitos Sexuais , Fixação de TecidosRESUMO
Most follicular lymphomas (FLs) have a phenotype consistent with the origin from CD5-, CD10+, bcl-6+ follicular center cells and can progress to diffuse large B-cell lymphoma (DLBCL). CD5 is expressed in about 10% of DLBCLs, showing prognostic value, whereas expression is rare in FL. We present 6 cases with coexisting features of CD5+ FL and CD5+ DLBCL, supporting a follicular origin for some CD5+ DLBCLs. The follicular areas showed a meshwork of CD21+ follicular dendritic cells that were lacking in the DLBCL areas. All cases showed a clonal CD19+, CD20+, CD5+, and CD10+ population in both follicular and diffuse areas. Molecularly, 4 of 6 cases demonstrated immunoglobulin heavy chain rearrangements and 1 case, a bcl-2/immunoglobulin heavy chain gene rearrangement. Somatic hypermutations were high in all 4 cases, in keeping with their germinal center origin. Four of five patients died of disease within 42 months, consistent with the proposed prognostic value of CD5 expression in DLBCL. Our data describe an aggressive variant of CD5+ FL suggesting the follicular origin of some CD5+ DLBCLs.
Assuntos
Biomarcadores Tumorais/metabolismo , Antígenos CD5/metabolismo , Linfoma de Células B/patologia , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/patologia , Idoso , Análise Mutacional de DNA , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
AIM: To investigate the seroprevalence of Helicobacter pylori (H pylori) infection and its more virulent strains as well as the correlation with the histologic features among patients who had undergone surgery for gastric cancer (GC). METHODS: Samples from 317 (184 males, 133 females, mean age 69+/-3.4 years) consecutive patients who had undergone surgery for gastric non-cardia adenocarcinoma were included in the study. Five hundred and fifty-five (294 males, 261 females, mean age 57.3+/-4.1 years) patients consecutively admitted to the Emergency Care Unit served as control. Histological examination of tumor, lymph nodes and other tissues obtained at the time of surgery represented the diagnostic "gold standard". An enzyme immunosorbent assay was used to detect serum anti-H pylori (IgG) antibodies and Western blotting technique was utilized to search for anti-CagA protein (IgG). RESULTS: Two hundred and sixty-one of three hundred and seventeen (82.3%) GC patients and 314/555 (56.5%) controls were seropositive for anti-H pylori (P<0.0001; OR, 3.58; 95%CI, 2.53-5.07). Out of the 317 cases, 267 (84.2%) were seropositive for anti-CagA antibody vs 100 out of 555 (18%) controls (P<0.0001; OR, 24.30; 95%CI, 16.5-35.9). There was no difference between the frequency of H pylori in intestinal type carcinoma (76.2%) and diffuse type cancer (78.8%). Intestinal metaplasia (IM) was more frequent but not significant in the intestinal type cancer (83.4% vs 75.2% in diffuse type and 72.5% in mixed type). Among the patients examined for IM, 39.8% had IM type I, 8.3% type II and 51.9% type III(type III vs others, P = 0.4). CONCLUSION: This study confirms a high seroprevalence of H pylori infection in patients suffering from gastric adenocarcinoma and provides further evidence that searching for CagA status over H pylori infection might confer additional benefit in identifying populations at greater risk for this tumor.
Assuntos
Adenocarcinoma/complicações , Infecções por Helicobacter/complicações , Infecções por Helicobacter/epidemiologia , Helicobacter pylori , Intestinos/patologia , Neoplasias Gástricas/complicações , Adenocarcinoma/etiologia , Adenocarcinoma/cirurgia , Idoso , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Estudos de Casos e Controles , Feminino , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Humanos , Itália/epidemiologia , Masculino , Metaplasia , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/cirurgiaRESUMO
This is the first description of an extensive immunohistochemical analysis of interferon (IFN)-inducible gene IFI16 expression in normal tissues. Immunohistochemical detection of IFI16 in paraffin-embedded tissues is achieved by using a polyclonal antibody raised against its C-terminal fragment that recognizes its three closely migrating isoforms in Western blotting. The results clearly indicate that IFI16 expression is not restricted to the hematopoietic compartment. In normal adult human tissues, it is prominent in stratified squamous epithelia and particularly intense in parabasal cells in the proliferating compartments, but it gradually decreases in the more differentiated suprabasal layers. Understanding of IFI16 expression in vivo is essential for interpretation of the results obtained from in vitro studies and elucidation of its physiologic role. The constitutive expression and wider distribution of IFI16 in normal human tissues, not restricted to the hematopoietic compartment, strongly support the possibility of an important role in cell differentiation that can be further modulated by other stimuli, such as IFN.
Assuntos
Células Epiteliais/metabolismo , Proteínas Nucleares , Fosfoproteínas , Proteínas/fisiologia , Western Blotting , Soluções Tampão , Diferenciação Celular/fisiologia , Divisão Celular , Ácido Cítrico , Sistema Digestório/citologia , Sistema Digestório/metabolismo , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas Imunoenzimáticas , Interferon gama/farmacologia , Masculino , Especificidade de Órgãos , Inclusão em Parafina , Biossíntese de Proteínas , Isoformas de Proteínas/análise , Proteínas/genética , Proteínas Recombinantes , Valores de Referência , Sistema Respiratório/citologia , Sistema Respiratório/metabolismo , Pele/citologia , Pele/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Fixação de Tecidos , Células U937 , Sistema Urogenital/citologia , Sistema Urogenital/metabolismoRESUMO
The expression of p53, c-erbB-2, bcl-2 and c-myc proteins was compared to the quantity of the nucleolar organiser regions (AgNORs) and MIB-1 antigen to elucidate the relationship between oncogene expression and rapidity of cell proliferation and tumor growth fraction. Sections from 50 male breast carcinomas (MBC) and 62 superficial papillary bladder neoplasias were stained with the standardised AgNOR method and monoclonal antibodies MIB-1, DO7, CB11, bcl-2 124 and 9E11. p53 immunopositivity was associated with high AgNOR quantity and MIB-1 scores both in MBC and bladder neoplasm. c-erbB-2 expression was associated with high AgNOR quantity in bladder neoplasm. bcl-2 expression was associated with low AgNOR quantity in MBC. c-myc expression was associated with high AgNOR quantity in MBC. MBC patients with low AgNOR quantity, and p53, c-erbB-2 and c-myc immunonegativity had the longest overall survival. Patients with bladder neoplasia with low AgNOR quantity, negative p53 and positive c-erbB-2 immunostaining had the longest disease-free survival time. Our results indicate that p53 overexpression reflects both the rapidity of cell proliferation, as assessed by AgNOR quantity, and tumor growth fraction, as assessed by MIB-1 scores, while c-erbB-2, c-myc and bcl-2 expression mainly reflects the rapidity of cell proliferation. The combination of AgNOR quantity and oncogene expression may stratify patients into different risk groups.
Assuntos
Neoplasias da Mama Masculina/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Antígeno Ki-67/biossíntese , Região Organizadora do Nucléolo/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/metabolismo , Divisão Celular , Núcleo Celular/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Oncogenes , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteína Supressora de Tumor p53/biossínteseRESUMO
OBJECTIVES: Tumor angiogenesis is an essential and complex process necessary for the growth of all tumors which represents a potential therapeutic target. Angiogenesis inhibitors targeting vascular endothelial growth factor (VEGF) or their receptor tyrosine kinases have been approved by the FDA. In thymic epithelial tumors (TET), targeted therapies have been sporadically applied due to their rarity. To ascertain the presence of potential therapeutic targets, we analyzed by immunohistochemistry the expression of angiogenesis-related biomarkers in a large series of TET arranged in Tissue Micro Arrays (TMA). MATERIALS AND METHODS: We assessed by immunohistochemistry the expression of the possible molecular target of anti-angiogenic therapy, i.e. VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, and PDGFRß, in a TMA series of 200 TET collected in the framework of a multi-institutional collaborative project for Rare Diseases. RESULTS: When compared to the low-risk tumors, high-risk TET (B2, B3, carcinomas) contained higher proportion of cancer cells expressing VEGFA, VEGFC and VEGFD (P<0.001, P<0.001, and P<0.001) growth factors, and their receptors VEGFR1 (P=0.002), VEGFR2 (P=0.013), and VEGFR3 (P=0.041). No differences were observed in terms of PDGFRß expression. CONCLUSIONS: According to our data, it is possible to hypothesize the existence of multiple paracrine and/or autocrine loops in TET, particularly in the high-risk ones, involved in TET growth and progression. Anti-angiogenic agents, directed to inhibit these loops, are therefore to be considered as potential tools in advanced TET therapy.
Assuntos
Neoplasias Epiteliais e Glandulares/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Neoplasias do Timo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores , Criança , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Epiteliais e Glandulares/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Estudos Retrospectivos , Neoplasias do Timo/tratamento farmacológico , Neoplasias do Timo/mortalidade , Neoplasias do Timo/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Adulto JovemRESUMO
Flow cytometry (FC) immunophenotyping of fine-needle aspiration (FNA) has been reported to be useful in the diagnosis of non-Hodgkin lymphomas (NHL). The authors reviewed their 5-year experience to assess the ability that FC has in improving the diagnostic capacity of cytomorphology in the diagnosis and subclassification of NHL according to the World Health Organization's classification. FC was performed on 252 FNA specimens. These included 123 cases of NHL (89 primary and 34 recurrent lymphomas). The FC immunophenotyping included CD3, CD4, CD8, CD10, CD19, CD20, CD45, and kappa/lambda antibodies combinations in the screening panel and additional panels for B or T lineage in the presence of positivity for lymphoma after the screening. An immunologic diagnosis was obtained by FC in 90% (111/123) of cases identified as NHL. FC was able to improve the total number of NHL detected in 8 cases where cytomorphology had failed to do so. In 7% (9/123) of cases, FC failed to formulate a diagnostic hypothesis owing to the sample inadequacy; 2 cases (2%) were not identified as lymphomas by FC (1 of them considered only "suggestive" also by cytomorphology); 1 case was not identified neither by FC, nor by cytomorphology. In cases having a histologic follow-up, levels of diagnostic sensitivity and specificity of the combination cytomorphology/FC were 97% and 94%, respectively. FC applied to FNA enhanced the diagnostic potential of cytologic diagnosis and subclassification of NHL, thus avoiding the need for invasive surgical biopsies in many cases.
Assuntos
Biópsia por Agulha Fina , Citometria de Fluxo , Imunofenotipagem , Linfoma não Hodgkin/diagnóstico , Linfoma não Hodgkin/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/citologia , Linfócitos B/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Linfoma não Hodgkin/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Linfócitos T/citologia , Linfócitos T/imunologia , Adulto JovemRESUMO
Intrasinusoidal infiltration (ISI) is a pattern of invasion that is rarely found on bone marrow (BM) biopsies, and is considered as a hallmark of splenic marginal zone cell lymphoma (SMZL). We analysed BM biopsies showing intrasinusoidal infiltration from 54 consecutive patients with different types of lymphoma to verify if ISI quantity was a diagnostic criterion for SMZL. There were 35 primary splenic lymphoma (PSL) and 19 non-PSL; 28 SMZL, three non-splenic MZL, six mantle cell, six small lymphocytic, four follicular, four diffuse large B cell, one peripheral T cell, one lymphoplasmacytic and one anaplastic large-cell lymphoma. The quantity of BM infiltrate was assessed on CD45, CD20 and CD3 stained sections. The mean percentage of total (TI) and intrasinusoidal (ISI) lymphocytes was calculated in 10 areas for each case. TI quantity was 21.57 in PSL and 35.05 in non-PSL (P = 0.04). ISI quantity was 5.23 in PSL and 7.62 in non-PSL (P = 0.08), 5.83 in SMZL and 2.83 in other types of PSL (P = 0.12), 4.46 in non-splenic MZL and 8.21 in other types of non-PSL (P = 0.28). No difference in ISI quantity was found among the lymphoma subtypes, either in PSL (P = 0.74) or non-PSL (P = 0.3). The data demonstrate that ISI quantity in BM biopsies is not a reliable diagnostic parameter for SMZL.
Assuntos
Neoplasias da Medula Óssea/secundário , Medula Óssea/patologia , Linfoma não Hodgkin/patologia , Neoplasias Esplênicas/patologia , Adulto , Idoso , Biópsia , Exame de Medula Óssea/métodos , Diagnóstico Diferencial , Feminino , Humanos , Linfoma não Hodgkin/diagnóstico , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Tamanho do Órgão , Neoplasias Esplênicas/diagnósticoRESUMO
Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.
Assuntos
Citocinas/metabolismo , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Neoplasias Pancreáticas/metabolismo , Fenótipo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Meios de Cultura , Citocinas/genética , Células Dendríticas/citologia , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , RNA Mensageiro/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic large-cell lymphomas (ALCLs) carry chromosome translocations in which the anaplastic lymphoma kinase (ALK) gene is fused to several partners, most frequently, the NPM1 gene. We have demonstrated that the constitutive activation of ALK fusion proteins results in cellular transformation and lymphoid neoplasia. Herein, we specifically down-regulated ALK protein expression by using small hairpin RNA (shRNA) targeting a sequence coding for the catalytic domain of ALK. The ablation of ALK leads to the down-modulation of known ALK downstream effectors, cell growth arrest, and reversion of the transformed phenotype of ALK(+) mouse embryonic fibroblasts in vitro and in vivo. In human ALCL cells lentiviral-mediated ALK knock-down leads to G(1) cell-cycle arrest and apoptosis in vitro and tumor growth inhibition and regression in vivo. Using a specific approach we have demonstrated that the survival and growth of ALK(+) ALCLs are strictly dependent on ALK activation and signaling. Therefore, ALK is a viable target for therapeutic intervention and its inactivation might represent a pivotal approach for the treatment of ALK lymphomas and other ALK-dependent human tumors.
Assuntos
Apoptose , Linfoma Anaplásico de Células Grandes/enzimologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Quinase do Linfoma Anaplásico , Animais , Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica , Fibroblastos , Imunofluorescência , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Camundongos Nus , Nucleofosmina , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases , Retroviridae/genética , TransfecçãoRESUMO
Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)-ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry-based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor-bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALK(K210R) or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína Substrato Associada a Crk/metabolismo , Citoesqueleto/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular , Movimento Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Imunoprecipitação , Linfoma Difuso de Grandes Células B/metabolismo , Espectrometria de Massas , Camundongos , Fosforilação , Receptores Proteína Tirosina Quinases , Células Tumorais CultivadasRESUMO
The protein expression of the cyclin-dependent kinase inhibitor p27 is often deregulated in human tumors. In lymphomas the inactivation of p27 is achieved through either increased degradation(1) or sequestration via D cyclins,(2) and p27 protein levels have been shown to have a prognostic significance.(1,3) Recently, S-phase kinase-associated protein 2 (Skp2) has been proved to mediate p27 degradation in normal cells(4-7) and to have oncogenetic properties.(8,9) In this study, B-, T-, and myeloid hematopoietic cell lines and a well-characterized panel of human lymphomas (n = 244) were studied for the expression of Skp2. In human lymphomas, the expression of Skp2 strongly related to the grade of malignancy, being low in indolent tumors and very high in aggressive lymphomas. Moreover, the percentages of Skp2- and S-phase-positive cells, as measured by DNA content or BrdU labeling, strictly matched and closely parallel that of Ki-67 and cyclin A. An inverse correlation between Skp2 and p27 was found in the majority of lymphoma subtypes. Nonetheless, most mantle cell lymphomas and a subset of diffuse large cell lymphomas failed to show this correlation, suggesting that alternative pathway(s) for the regulation of p27 might exist. The detection of Skp2 protein either by flow cytometry or by immunohistochemistry represents a simple method to precisely assess the S phase of lymphomas. The potential diagnostic and prognostic value of Skp2 is discussed.