The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [85]-98).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 46-69).

Waldenström's Macroglobulinemia (WM) is a rare, indolent, non-Hodgkin's lymphoma whose molecular pathology remains poorly understood. This disease is characterized by the accumulation of IgM-secreting lymphoplasmacytic cells in the bone marrow, and is often histopathologically indistinguishable from marginal zone lymphoma, IgM-secreting myeloma, and chronic lymphocytic leukemia with plasmacytic differentiation. To better understand the genomic landscape of this disease, whole genome sequencing was performed on bone marrow samples from thirty WM patients, ten of which were paired with germline tissue. This study identified two genes that are frequently mutated in WM: MYD88 and CXCR4. MYD88 was somatically mutated in 90% of WM samples, which displayed a single nucleotide variant resulting in a leucine to proline substitution at position 265. As prev iously demonstrated in activated B-cell subtype of diffuse large B-cell lymphoma, this mutation results in constitutive activation of the Toll-like receptor pathway and activation of nuclear factor kappa B (NF𝜅B). Highly sensitive allele specific polymerase chain reaction assays were developed to detect MYD88L265P in WM and related hematological malignancies. These studies demonstrated that MYD88L265P could be used to aid in the differential diagnosis, response assessment, and detection of minimal residual disease in WM. Moreover, MYD88L265P was observed in 50% of the precursor condition, IgM monoclonal gammopathy of undetermined significance, suggesting that it is an early event in the pathogenesis of WM. Blocking MYD88 dimerization or the use of downstream IRAK1/4 kinase inhibitors decreased the phosphorylation and nuclear localization of NF𝜅B. Somatic mutations in CXCR4 were only found in the regulatory C-terminal tail and were present in 29% of WM patients. These mutations were similar or identical to those found in the germline of patients with the autosomal dominant disease Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome. CXCR4 somatic WHIM-like mutations were found nearly exclusively in MYD88L265P mutated patients. These mutations impaired receptor internalization, increased signaling downstream of CXCR4, and instilled resistance to several WM directed therapeutics. WM patients who were wild type for both CXCR4 and MYD88 demonstrated inferior overall survival. These studies evidence highly recurring somatic events, and provide a genomic basis for the molecular pathogenesis of WM.

Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2004Bitstream added on 2014-06-13T20:36:39Z : No. of bitstreams: 1 silva_gn_me_botfm.pdf: 467036 bytes, checksum: 3b9fa526eae390b7ae86bf47c5bcf731 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O linfoma de Hodgkin (LH) tem características clínicas e anátomo-patológicas distintas dos linfomas não-Hodgkin. Seu componente neoplásico, as células células de Hodgkin/Reed-Sternberg (H-RS), corresponde a cerca de 2% do tumor. As células H-RS apresentam imunofenótipo peculiar e sua origem ainda é objeto de estudo. O LH é classificado em LH clássico (que inclui os subtipos celularidade mista, esclerose nodular, depleção linfocítica e LH rico em linfócitos) e LH predominância linfocítica nodular. A associação do vírus de Epstein-Barr (EBV) com LH é conhecida e acredita-se que este vírus desempenha importante papel no desenvolvimento de parcela significativa dos casos de LH. No LH, o acúmulo de células reativas como linfócitos, plasmócitos, eosinófilos, histiócitos e fibroblastos ocorre em resposta a citocinas secretadas pelas células H-RS. A interleucina 10 (IL-10), importante citocina antiinflamatória da resposta imunitária, tem sido encontrada em grande quantidade em indivíduos com LH. É possível que essa elevada expressão de IL-10 esteja vinculada a determinados polimorfismos de nucleotídeos simples (SNP) na seqüência promotora do gene da IL-10, que podem se associar a características anátomoclínicas do LH. O presente trabalho avaliou a freqüência dos polimorfismos da IL-10 nas posições promotoras -1082, -819/-592 e estudou a correlação destes polimorfismos em LH com subtipos histológicos, infecção pelo EBV, faixa etária e, em alguns casos, estadiamento clínico da doença. Os resultados demonstram que os diferentes fenótipos para produção de IL-10, genótipos na posição -1082 e genótipos nas posições -592/-819 não têm relação com subtipos do LH e idade dos pacientes. Por outro lado, verificou-se que o genótipo GG na posição -1082 e a combinação de haplótipos GCC/GCC para alta... / Clinical and pathologic features of Hodgkin’s lymphoma (HL) reflect an abnormal immune response, which is in part due to the elaboration of a variety of cytokines. It was reported that interleukin 10 (IL -10), which may be produced by the malignant Hodgkin/Reed-Sternberg (H-RS) cells, is an important prognostic factor in HL, and it could play a role in the pathogenesis of this neoplasm. It is well established that genetic factors affect protein expression and function. In this regard, polymorphisms in the promoter region of IL-10 gene may cause different phenotypes for IL- 10 synthesis and activity. Three dimorphic single nucleotide polymorphisms (SNPs) have been identified at positions -1082, -819 and -519 within the IL-10 promoter region (SNPs/IL-10). These polymorphisms are in close proximity to several transcription factors binding-sites, and may interfere with IL-10 gene transcription. The aim of this study was to evaluate whether is there a particular distribution of SNPs at positions -1082, -819 and -519 in the IL-10 gene promoter in patients with HL, as well as to access the differences in the SNPs/IL10 frequency regarding HL subtype, patient age, EBV infection status, and clinical staging of the disease. For these purposes, sixty-five cases of HL and fifty cases of reactive follicular lymphoid hyperplasia (RFLH) were evaluated for SNPs/IL-10 by polymerase chain reaction amplification plus restriction fragment length polymorphism analysis (PCR-RFLP). Compared to HL EBV-negative cases, in HL EBV-positive cases it was observed a significant increase in the frequency of GG genotype at position -1082 of IL-10 gene promoter region, which is associated to high level of IL -10 production. No differences were observed in the frequency of different SNPs/IL-10 concerning patient age, HL subtype, and clinical stage of the disease. These results... (Complete abstract, click electronic address below)

Resumo: O Linfoma de Hodgkin (LH) é uma neoplasia linfóide histologicamente caracterizada pela presença de escassas células neoplásicas peculiares, denominadas células de Hodgkin/Reed-Sternberg (H-RS), em meio a infiltrado inflamatório não-neoplásico. Seu curso clínico está diretamente relacionado ao estadiamento, subtipo histológico e presença de alguns fatores prognósticos adversos já estabelecidos. Sintomas constitucionais como febre, perda de peso e sudoreses noturna, associados a um aparente desbalanço da resposta imune celular, são características comuns ao diagnóstico e denotam anormalidade da resposta imunológica desses pacientes, provavelmente causada pela produção de diferentes citocinas, tanto pelas células H-RS como pelo infiltrado inflamatório de permeio. Na maior parte dos casos de LH, há produção aumentada de citocinas de padrão Th2. No presente estudo, foram determinadas, por ELISA, as concentrações séricas de IL-6, IL-10 e IL-13 de 28 pacientes com LH clássico, antes e depois do tratamento anti-neoplásico, e de 26 voluntários saudáveis que compuseram grupo-controle. Dosagens de IL-6 e IL-10 pré-tratamento foram significativamente maiores nos pacientes em relação ao grupo-controle. Todos os pacientes apresentaram redução do nível sérico de IL-6 e IL-10 após o tratamento antineoplásico. Ao diagnóstico, níveis elevados de IL-6 foram associados à presença de linfonodomegalia abdominal, hepatomegalia, sintomas B e anemia. O estudo da regressão linear múltipla mostrou que sintomas B e linfocitopenia são bons preditores de níveis séricos pré-tratamento de IL-6. Níveis elevados de IL-10 foram relacionados à hipoalbuminemia e hepatomegalia. Nenhum paciente apresentou dosagem Resumo 52 sérica detectável de IL-13. Os resultados demonstram que níveis séricos elevados de IL-6 e IL-10 ao diagnóstico estão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hodgkin's lymphoma (HL) is a malignant lymphoid neoplasia characterized by abnormal immune response. Part of this disturbance is attributable to the activity of cytokines produced by the malignant Hodgkin/Reed-Sternbergh cells and reactive inflammatory cells. IL-6, IL-10 and IL-13 seem to play an important role in the pathogenesis of HL. Although some features of the disease have been associated with the production of these interleukins, there is insufficient data about changes in its serum levels at diagnosis and after the treatment, as well as its potencial use as biomarkers of HL course. Design and Methods. Serum levels pre and post treatment of IL-6, IL-10 and IL-13 in 28 patients with HL were determined by ELISA. Results were evaluated against clinical and laboratory parameters, as well as response to treatment and presence of infection by the Epstein-Barr virus (EBV), assessed by in situ hybridization with biotinylated probe directed to the viral transcript EBER-1. Serum samples from 26 healthy blood-donors volunteers were evaluated as a control group. Results. IL-6 and IL-10 serum levels before treatment of HL were significantly higher in patients compared to healthy individuals (p<0,001), and a significant reduction after treatment of the disease was observed (p<0,001). Serum levels of IL-13 were indetectable both in patients (before and after treatment) and controls. Serum levels of IL-6 before treatment were higher in patients with abdominal involvement by HL (p=0,03), hepatomegaly (p=0,04), B-symptoms (p=0,01), and anemia (p=0,02). On the other hand, IL-10 pre-treatment levels were higher in patients with hypoalbuminemia (p=0,04) and hepatomegaly (p=0,01). Multivariate analysis Abstract 54 revealed that lymphocytopenia and B-symptoms were good predictors of IL-6 levels in serum before treatment of patients... (Complete abstract click electronic access below) / Orientador: Deilson Elgui de Oliveira / Coorientador: Lígia Niéro-Melo / Banca: José Salvador Rodrigues de Oliveira / Banca: Maria Claudia Nogueira Zerbini / Mestre

Orientador: Jose Vasallo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T18:25:59Z (GMT). No. of bitstreams: 1 Lira_MarianaMontenegrodeMelo_D.pdf: 3297423 bytes, checksum: 565895365a98b6df746479d5e2d8074c (MD5) Previous issue date: 2008 / Resumo: A micose fungóide (MF) corresponde a cerca de 50% de todos os linfomas primários da pele. Trata-se de uma neoplasia epidermotrópica constituída por linfócitos atípicos, com fenótipo predominantemente de linfócito T CD4+. O diagnóstico da MF é freqüentemente muito difícil, tanto pela sobreposição com dermatoses benignas, como pela discordância entre achados clínicos e histopatológicos. Embora a certeza de malignidade repouse na observação do comportamento clínico, parâmetros histopatológicos, morfométricos e imunofenotípicos têm sido utilizados para indicar malignidade. Em estudos prévios realizados pelo nosso grupo, não confirmamos os achados de alguns autores, que enfatizam que níveis elevados da relação CD4:CD8 sugerem malignidade. Entretanto, tivemos a oportunidade de comprovar a utilidade da perda da expressão do marcador linfóide T, CD7, no diagnóstico das lesões linfomatosas. Neste último estudo, ficou-nos a impressão de que a ausência de expressão do CD7 era mais evidente em células linfóides mais volumosas e atípicas. Dando continuidade a esta linha de investigação, os objetivos do presente trabalho são os de determinar o papel da avaliação morfométrica em tecidos incluídos em parafina e submetidos à reação imunoistoquímica com CD3 na diferenciação das infiltrações linfocitárias cutâneas e determinar se existe correlação entre o aumento da área nuclear e o percentual de expressão do CD7 nestas lesões. Foram estudados 30 casos de MF e 11 casos de infiltrados linfóides cutâneos benignos. Imagens de 100 células positivas de cada caso foram capturadas e analisadas utilizando o sistema de análise de imagens Imagelab®, sendo mensurados perímetro (P), área (A) e diâmetros (D) nucleares e calculado o índice de contorno nuclear (ICN= P/?A). Houve diferença estatisticamente significante entre as médias de todas as variáveis (p< 0,05), sendo A a variável com maior poder de discriminação entre os grupos (r² = 80,82%). E observou-se uma tendência de correlação entre a perda de expressão de CD7 e o aumento da área nuclear. Ficou, assim, demonstrado que a avaliação de parâmetros nucleares de células previamente identificadas como linfócitos T através da imunocoloração para o CD3 permite a diferenciação morfométrica entre MF e infiltrações linfocitárias benignas. Este método adiciona critérios objetivos para auxiliar no diagnóstico da MF / Abstract: Mycosis fungoides (MF) represents the most common skin lymphoid neoplasm. In initial stages, differential diagnosis of MF with other benign dermal lymphoid infiltrates (BDLI) may be impossible on morphological basis alone. In previous studies conducted by our group, only loss of immunoexpression of CD7 in MF proved to be of diagnostic help, but not the ratio between immunoexpression of CD4 and CD8. In the present study, 30 cases of MF and 11 cases of BDLI were analyzed, in order to compare morphometric parameters, which could be of diagnostic aid. As CD7 is frequently deleted in MF, immunohistochemical detection of T-cells was made using an antibody to CD3. Images of 100 CD3-positive cells per case in both groups were captured and analyzed using a simple computer program (Imagelab®) for nuclear perimeter (P), area (A), diameters (D) and nuclear contour index (NCI). All parameters showed statistically significant higher values for MF. Area was the variable with the strongest discriminating power between the two groups of patients. There was a tendency to a correlation between the loss of expression of CD7 and increase in nuclear area. Even if morphologic evaluation is not accurate to distinguish benign versus malignant dermal lymphoid infiltrates, due to the variability of size and shape of these cells, a more sensitive method promptly shows this difference. Our results suggest that morphometry of CD3-positive lymphoid cells may add valuable information in the differential diagnosis of MF and benign dermatoses / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas

No description available.

Abstract Gelatinases (MMP-2 and MMP-9) play a key role during invasion and metastazising of malignant cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumours. However little is known about their role in hematological malignancies. In the present work, gelatinase expression and its clinicopathological correlations were studied with immunohistochemical staining in 10 cases representing normal bone marrow aspirate smears, 123 cases representing diagnostic bone marrow samples of patients with different leukaemias (35 AML, 7 CLL, 6 CML, 75 ALL), 67 diagnostic paraffin-embedded lymph node biopsies from patients with Hodgkin's lymphoma and 57 biopsies from patients with non-Hodgkin's lymphomas. The lymphoma samples were also stained with factor VIII antibody to evaluate the extent of new vessel formation and the non-Hodgkin's lymphoma cases also with tissue inhibitor of metalloproteinases -1 (TIMP-1) antibody. CLL did not express either of the MMP enzymes, while CML in the chronic phase expressed strongly both of the enzymes. In ALL, gelatinase expression was weak and detectable in pediatric cases in only 12.7% and in the adults in 65% of the cases. In adult ALL, MMP-2 expression correlated strongly with an extramedullary and invasive pattern of disease presentation. In AML MMP-2 positivity had markedly favorable prognostic and predictive power. In lymphoma studies, no correlations could be detected between gelatinase expression and the clinical parameters of invasion. MMP-9 positivity was related to the presence of B symptoms, which difference was statistically significant in Hodgkin's lymphoma. In Hodgkin's lymphoma, strong MMP-9 expression also implicated decreased neovascularization. In both lymphoma types, strong MMP-9 expression correlated with unfavorable prognosis, which difference was statistically significant in non-Hodgkin's lymphomas and remained as a tendency in Hodgkin's lymphoma. MMP-2 had statistically significant association with a favorable prognosis in Hodgkin's lymphoma. Combination of the results of both stainings further increased prognostic power. All together these findings implicate that gelatinases could be used as prognostic tools in AML and lymphomas albeit this needs to be verified in larger materials.