Dissertations / Theses on the topic 'Hematologic neoplasm'

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Mestrado Acadêmico em Ciências do Cuidado em Saúde
Trata-se de uma pesquisa qualitativa, descritiva do tipo estudo de caso único, que teve como objeto de estudo cuidados de enfermagem aos pacientes onco-hematológicos hospitalizados e os seguintes objetivos: caracterizar o perfil dos pacientes onco-hematológicos hospitalizados na enfermaria de hematologia do Hospital Universitário Antônio Pedro (HUAP), identificar as demandas de cuidado dos pacientes onco-hematológicos hospitalizados na enfermaria de hematologia do HUAP, descrever os cuidados realizados pela equipe de enfermagem aos pacientes onco-hematológicos hospitalizados na enfermaria de hematologia do HUAP, elaborar um protocolo de cuidados de enfermagem aos pacientes onco-hematológicos hospitalizados na enfermaria de hematologia do HUAP, a partir dos relatos dos membros da equipe de enfermagem. O cenário de pesquisa foi à unidade de hematologia do HUAP. Fizeram parte do estudo 17 sujeitos, sendo sete (7) pacientes hospitalizados com diagnóstico médico de LMA, LMC, LLA, LLC, Linfoma de Hodgkin e Não-Hodgkin e dez (10) membros da equipe de enfermagem. A coleta de dados foi através do formulário de identificação e o histórico de enfermagem com os pacientes e a observação participante acerca do cuidado de enfermagem ao paciente onco-hematológico hospitalizado e as entrevistas com a equipe de enfermagem. Os resultados apontaram como demandas de cuidado de enfermagem àqueles relacionados à atenção, cuidados gerais, ambiente de cuidado; orientação à família e ao paciente e equipe especializada. Com relação ao cuidado que a equipe de enfermagem realiza foram identificados: cuidado prevenção; cuidado sensível; cuidado clínico; cuidado atributos; cuidado educativo. E o cuidado específico ao paciente onco-hematológico está atrelado à prevenção, conforto, apoio emocional e orientação e tais cuidados foram os subsídios para a elaboração do protocolo de cuidado de enfermagem a estes pacientes segundo a realidade local do HUAP. Desta forma, tal protocolo consolida o que a enfermagem do serviço de hematologia do HUAP já vem realizando. Além disso, torna-se aos membros desta equipe um apoio na prestação da assistência de enfermagem de modo que possam identificar no protocolo os cuidados específicos direcionados as demandas de cuidados destes pacientes.
This is a qualitative, descriptive research of type single case study, which had as its object of study nursing care to onco-hematological patients hospitalized and the following goals: characterize the profile of onco-hematological patients hospitalized in hematology ward of the University Hospital Antônio Pedro (HUAP), identifying the demands of care of oncohematological patients hospitalized in hematology ward of HUAP, describing the care provided by nursing staff to onco-hematological patients hospitalized in hematology ward of HUAP, draw up a protocol of nursing care to onco-hematological patients hospitalized in hematology ward of HUAP, from reports of the members of the nursing staff. The scenario of research was the Hematology unit of HUAP. Were part of the study 17 subjects, being seven (7) hospitalized patients with medical diagnosis of LMA, LMC, LLA, LLC, Hodgkin's lymphoma and non-Hodgkin lymphoma and ten (10) members of the nursing staff. The data collection was through the identification form and the nursing history with patients and the participant observation about nursing care to the onco-hematological patient hospital and interviews with the nursing staff. The results showed how nursing care as demands related to attention, general care, environment of care; guidance to the family and the patient and expert staff. With respect to the care that nursing staff conducts were identified: preventive care; sensitive care; clinical care; careful attributes; educational care. And patient specific oncohematological care is related to prevention, comfort, emotional support and guidance and such care was the allowances for nursing care protocol to these patients according to the local reality of HUAP. In this way, such a protocol that consolidates what the Hematology nursing service of HUAP already has been performing. Moreover, it becomes to members of this team a support in the provision of nursing care so that they can identify the protocol specific targeted care demands these patients.

As anormalidades hematológicas de origem paraneoplásica são identificadas em diversos tipos de neoplasias que acometem cães e gatos. Nas neoplasias mamárias em cadelas, já foram identificadas anormalidades relacionadas com a coagulação, onde verificou-se que a coagulação intravascular disseminada (CID) clínica e subclínica pode estar presente em 83% das cadelas com carcinoma mamário. Na medicina humana, é dada relevância à investigação de tais alterações uma vez que são fatores indicadores de prognóstico do câncer. Enquanto isso, na medicina veterinária, são escassos os estudos que relacionam as alterações hematológicas com o tipo tumoral, estadiamento e determinação de prognóstico. O objetivo do presente estudo foi realizar a avaliação hematológica, bioquímica e da hemostasia de cadelas acometidas por neoplasia mamária para identificar a alteração mais frequente, além de relacionar as anormalidades com o estadiamento tumoral. Para isso, foram utilizadas 25 cadelas atendidas pelo Grupo de Estudos em Oncologia em pequenos animais (ONCOVET) do Hospital de Clínicas Veterinárias da UFRGS (HCV-UFRGS) durante o período de 4 meses. Foi realizado coleta de sangue para hemograma, contagem de plaquetas, bioquímica sérica (albumina, ALT, cálcio, creatinina, FA, glicose, ureia) e teste de coagulação que constou de TP (tempo de protrombina), TTPa (tempo de tromboplastina parcial ativada), TT (tempo de trombina), fibrinogênio e mensuração do dímero-D. O estadiamento tumoral foi obtido através do exame físico e do resultado da biopsia das mamas. As anormalidades encontradas incluíram anemia, leucocitose neutrofílica, monocitose, eosinofilia, trombocitose, hipoalbuminemia, hipocalcemia, hipoglicemia e diminuição dos níveis de ureia sanguínea. Entretanto, essas alterações não foram relacionadas diretamente com a progressão tumoral, uma vez que não houve diferença entre os grupos avaliados. Apenas as variáveis RDW e ALT apresentaram relação significativa entre os grupos, contudo, sem relevância clínica. No teste de coagulação, houve diferença significativa entre os grupos apenas no TT e fibrinogênio, que foi relacionado com o estadiamento tumoral.
Hematological abnormalities of paraneoplastic origin are identified in several types of cancers that affect dogs and cats. In dogs with mammary neoplasms, abnormalities associated with coagulation have been identified, and verified that disseminated intravascular coagulation (DIC) clinical and subclinical may be present in 83% of dogs with mammary carcinoma. In human medicine, research in this field has been relevant since those factors are indicators of cancer prognosis. Meanwhile, in veterinary medicine, there are few studies that relate hematological changes with tumor type, staging and determination of prognosis. The aim of this study was to evaluate the hematological, biochemical and hemostathic abnormalities in bitches affected by mammary cancer to identify the most frequent alteration and associate with tumor staging. For this, 25 bitches attended by the Oncology Study Group in small animals (ONCOVET) of the Veterinary Hospital of UFRGS (HCV-UFRGS) for the period of 4 months were used. Blood collection for complete blood count, platelet count, serum biochemistry (albumin, ALT, calcium, creatinine, ALP, glucose, urea) and coagulation test that consisted of PT (prothrombin time), aPTT (activated partial thromboplastin time), TT (thrombin time), fibrinogen and D-dimer measurement were performed. Tumor staging was obtained by physical examination and the results of the biopsy of the breast. The abnormalities found included anemia, neutrophilic leukocytosis, monocytosis, eosinophilia, thrombocytosis, hypoalbuminemia, hypocalcemia, hypoglycemia and decreased levels of urea. However, these changes were not associated directly with tumor progression, since there was no difference among the groups. Only the RDW and ALT variables was associated significantly between the groups, however, with no clinical relevance. In the coagulation test, there was significant difference between the groups only in TT and fibrinogen, which was associated with tumor staging.

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

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Fundação para o Desenvolvimento Médico e Hospitalar (Famesp)
O processo de adoecer provoca uma crise vital para a pessoa. Na criança traz repercussões mais sérias, pois os conteúdos do inconsciente estão ainda pouco discriminados pelo ego e nesta situação pela percepção da possibilidade de desintegração ou morte, alguns destes conteúdos tornou-se mais intrusos e ameaçadores. Enfermidades graves durante a infância ou na adolescência tais como, as neoplasias, doenças cardíacas, insuficiência renal, diabetes, doenças com má formação genéticas, doenças hematológicas, dentre outras, conduzem o paciente e seus familiares a situações de crise, manifesta por estresse emocional e comportamental, perturbações da auto-imagem e auto-estima, comprometimento da qualidade de vida, dentre outros sintomas psicopatológicos. O objetivo deste trabalho foi o de avaliar a intervenção psicoterápica em modelo analítico em crianças e adolescentes com doenças onco-hematológicas. Foram utilizados dois instrumentos de avaliação: inventários de estresse e depressão infantil (ISI e CDI) aplicados em 4 diferentes momentos após a primeira avaliação: TO, T30, T60 , T90 e T120. Foram acompanhados 7 pacientes do ambulatório de psicologia OEI do Hemocentro de Marília. Trata-se de um ensaio clínico não-randomizado intra-grupos de séries temporais, no qual a intervenção foi realizada com um único grupo e os desfechos foram comparados em cada participante durante diferentes períodos desta intervenção. A análise dos escores CDI ao final do período das intervenções observou-se uma melhora no estado geral dos sintomas de depressão dos participantes (escores finais aos 120 dias inferiores a 17) comprovando a eficácia da intervenção psicoterápica de base analítica na melhora dos sintomas em crianças e adolescentes portadores de neoplasias onco-hematológicos. Os dados...
The process of getting sick causes a vital crisis to the person. It brings more severe repercussions to the children since the unconscious contents are hardly discriminates by the ego and, in this special situation, by the perception of the possibility of disintegration or even death. So some of these contents become more threatening. Serious diseases during childhood or adolescence such as neoplasi, cardio illness, kidney insufficiency, diabetes, genetics and blood problems, among others, lead the patient and his family members to crises with emotional and behavior stress, self-esteem disturbance, even involving the daily life and other psychopathic symptoms. The objective on this paper was to evaluate a psychotherapeutic intervention in an analytical pattern for children and teenagers with onco-hemathology diseases. Two models for evaluation were used: Stress inventory and depression on in children (ISI and CDI) applied in four different times after the first evaluation: TO, T30, T60, T90 and T12. Seven patients of the psychology ambulatory OEI in the Hemocentro in Marilia were watched. It is a clinic essay not at random among groups on specific times in which the intervention was accomplished with a unique group and the results were compared on each participant in different times. In the analyses of the scores CDI in the end of interventions, some improvements in the overall depression symptoms were clearly noticed (final scores in 120 days bellow 17) proving the effectiveness of the psychotherapeutic intervention based on the analysis of the symptoms in kids and teen suffering from neoplasi and oncohematology diseases. The inventory data inn children stress are similar to the depression pointing to the effectives of the psychotherapeutic approach. The variations of the physical reactions (ESI) are related to the... (Complete abstract click electronic access below)

Orientadores: Maria Luiza Moretti, Angélica Zaninelli Schreiber
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: os fungos do gênero Fusarium são ubíquos no meio ambiente e causam doenças em plantas e aos seres humanos. Objetivos: 1)estabelecer o protocolo para a coleta e o isolamento de Fusarium spp. no ar; 2)isolar fungos do gênero Fusarium do ar da enfermaria de Hematologia e da Unidade de Transplante de Medula Óssea (TMO) no Hospital de Clínicas (HC) da Universidade Estadual de Campinas (UNICAMP) e comparar a relação genética de isolados ambientais de Fusarium spp. com os isolados a partir de hemoculturas de rotina diagnóstica de pacientes hospitalizados durante os anos de 2002 a 2013, 3)identificar os fungos do gênero Fusarium ao nível de complexo de espécies por PCR em tempo real, e ao nível de espécie pelo sequenciamento de fragmento do gene EF1?. Metodologia: foram colhidas amostras de ar nas unidades de TMO (que possui controle de ar através de filtros HEPA e fluxo de ar de pressão positiva) e na Hematologia (unidade sem controle do ar) do HC. De 2006 a 2013 foram identificados 18 isolados de Fusarium spp. em culturas de sangue. A coleta de ar foi realizada através do amostrador de ar BioSamp modelo MBS 1000D (YotsubishiCorp Japão). Um meio de cultura seletivo para Fusarium (Agar Komada com modificações) foi usado na placa de Petri dentro do amostrador de ar. O volume de ar de 1000 mL e 500 mL foram aspirados da Hematologia e enfermarias de TMO, respectivamente. Após o tempo de incubação, as colônias que cresceram nas placas foram avaliadas macroscopicamente e microscopicamente para identificação morfológica. Fusarium spp. foram identificados através de um novo ensaio de PCR em tempo real composto por dois ensaios de PCR em tempo real: um específico para o gênero Fusarium e um ensaio específico para detecção específica do complexo de espécies de Fusarium solani (FSSC). Para confirmar o complexo de espécies de Fusarium, uma região parcial do gene EF-1? foi sequenciado. O alinhamento das sequências foi realizado no programa Clustal W e a análise filogenética utilizou o programa Mega5 com o algorítimo Neighbor-Joininge Bootstrap de 1000 replicatas. Resultado: foram isoladas 108 cepas de Fusarium spp. destes 28% na unidade de TMO e 78% na Hematologia. Os complexos de espécies encontrados foram: 10% FCSC, 21% FIESC, 1% FOSC, 12% FSSC, 51% GFSC e 5% não definido. Na TMO a maior prevalência foi da espécie Fusarium solani (9/30 isolados) e na Hematologia Fusarium verticillioides (25/31 isolados). Não foi possível constatar relevância entre a temperatura e umidade do ar em relação ao aumento ou diminuição de isolados nas coletas. Nove isolados FSSC do ar e 8 de sangue apresentaram BT 99%. Conclusão. Os dados de análise filogenética sugeriram que os isolados de sangue de pacientes e ambientais foram similares sugerindo que o ambiente pôde representar uma fonte potencial de infecção para os pacientes imunodeprimidos
Abstract: Introduction:the fungus Fusarium are ubiquitous in the environment and cause diseases in plants and humans. Objectives: isolate fungi Fusarium in the air of the ward Hematology and Unit of Bone Marrow Transplantation (BMT) at the Hospital de Clinicas (HC), State University of Campinas (UNICAMP), compare the phylogenetic relationship of environmental isolates of Fusarium spp .with isolates from blood cultures of routine diagnosis of hospitalized patients during the years 2002-2013, to establish protocol for the collection and isolation of Fusarium spp. in the air, identify the fungi Fusarium species complex level by real time PCR, and to species level by sequencing the fragment EF1 ? gene. Methodology: air sample was collected in BMT units (which has control of air through HEPA filters and air flow positive pressure) and Hematology (there is not self control air inlet) HC. From 2006 to 2013, 18 samples obtained from blood cultures with the presence of Fusarium spp. were added to study the relationship between degree of molecular clinical isolates isolated from the air. The air sampling was performed using the Bio air sampler Samp MBS model 1000D (Yotsubishi Corp. Japan). A selective medium for Fusarium (Komada Agar changes by Y. Mikami, Chiba University) was used in the petri dish in the air sampler. The air volume of 1000 mL and 500 mL were aspirated Hematology and BMT wards, respectively. After the incubation time, colonies that grew on the plates were evaluated macroscopically and microscopically for morphological identification. Fusarium spp. were identified by PCR of a new system in real time according to Muraosa et al. comprising two PCR assays in real time: the specific assay Fusarium and a specific assay for specific detection of Fusarium solani (FSSC) complex. To confirm the complex of Fusarium species, a partial region of the EF-1? gene was sequenced. The alignment of sequences was performed in Clustal W program and phylogenetic analysis used Mega5 program with the neighbor-joining algorithm and bootstrap 1000 replicates. Results: 108 ceepas Fusarium spp were isolated. 28% of these isolates form the BMT unit and 78% in Hematology. Overall the species complexes ficarm divided as follows: 10% FCSC, 21% FIESC 1% FOSC, FSSC 12%, 51% and 5% GFSC nd not defined). In the BMT was higher prevalence of the species Fusariu solani (9/30 isolates) and the Hematology espéciecom largest presence is Fusarium verticillioides (25/31 isolates). Unable to find relevance between temperature and air humidity in relation to the increase or decrease in collections of isolates. Nine isolates of Fusarium solani air obtained from the same source ancestras 8 Fusarium solani isolates from blood (BT 99%). Conclusion:Fusarium spp were isolated in 108 environmental samples en all evaluated environments. Phylogenetically findings suggest equivalence between environmental isolates and isolates from the blood of patients. The collection protocol set showed satisfactory for the specific isolation of Fusarium result. The findings of Fusarium were grouped into complexes FSSC and FSSC not the technique of real-time PCR and sequencing of the gene EF1? allowed to classify the isolates in the species level
Mestrado
Clinica Medica
Mestre em Ciências

Orientador: Elenice Deffune
Banca: Roseli Nunes Antunes
Banca: Juliana Bassalobre Carvalho Borges
Resumo: O processo de adoecer provoca uma crise vital para a pessoa. Na criança traz repercussões mais sérias, pois os conteúdos do inconsciente estão ainda pouco discriminados pelo ego e nesta situação pela percepção da possibilidade de desintegração ou morte, alguns destes conteúdos tornou-se mais intrusos e ameaçadores. Enfermidades graves durante a infância ou na adolescência tais como, as neoplasias, doenças cardíacas, insuficiência renal, diabetes, doenças com má formação genéticas, doenças hematológicas, dentre outras, conduzem o paciente e seus familiares a situações de crise, manifesta por estresse emocional e comportamental, perturbações da auto-imagem e auto-estima, comprometimento da qualidade de vida, dentre outros sintomas psicopatológicos. O objetivo deste trabalho foi o de avaliar a intervenção psicoterápica em modelo analítico em crianças e adolescentes com doenças onco-hematológicas. Foram utilizados dois instrumentos de avaliação: inventários de estresse e depressão infantil (ISI e CDI) aplicados em 4 diferentes momentos após a primeira avaliação: TO, T30, T60 , T90 e T120. Foram acompanhados 7 pacientes do ambulatório de psicologia OEI do Hemocentro de Marília. Trata-se de um ensaio clínico não-randomizado intra-grupos de séries temporais, no qual a intervenção foi realizada com um único grupo e os desfechos foram comparados em cada participante durante diferentes períodos desta intervenção. A análise dos escores CDI ao final do período das intervenções observou-se uma melhora no estado geral dos sintomas de depressão dos participantes (escores finais aos 120 dias inferiores a 17) comprovando a eficácia da intervenção psicoterápica de base analítica na melhora dos sintomas em crianças e adolescentes portadores de neoplasias onco-hematológicos. Os dados... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The process of getting sick causes a vital crisis to the person. It brings more severe repercussions to the children since the unconscious contents are hardly discriminates by the ego and, in this special situation, by the perception of the possibility of disintegration or even death. So some of these contents become more threatening. Serious diseases during childhood or adolescence such as neoplasi, cardio illness, kidney insufficiency, diabetes, genetics and blood problems, among others, lead the patient and his family members to crises with emotional and behavior stress, self-esteem disturbance, even involving the daily life and other psychopathic symptoms. The objective on this paper was to evaluate a psychotherapeutic intervention in an analytical pattern for children and teenagers with onco-hemathology diseases. Two models for evaluation were used: Stress inventory and depression on in children (ISI and CDI) applied in four different times after the first evaluation: TO, T30, T60, T90 and T12. Seven patients of the psychology ambulatory OEI in the Hemocentro in Marilia were watched. It is a clinic essay not at random among groups on specific times in which the intervention was accomplished with a unique group and the results were compared on each participant in different times. In the analyses of the scores CDI in the end of interventions, some improvements in the overall depression symptoms were clearly noticed (final scores in 120 days bellow 17) proving the effectiveness of the psychotherapeutic intervention based on the analysis of the symptoms in kids and teen suffering from neoplasi and oncohematology diseases. The inventory data inn children stress are similar to the depression pointing to the effectives of the psychotherapeutic approach. The variations of the physical reactions (ESI) are related to the... (Complete abstract click electronic access below)
Mestre

A pessoa com distúrbios hematológicos demonstra desarranjos físicos, emocionais e sociais. Assim, atuar nesta área é estar disposta a cuidar do indivíduo, percebendo e intervindo precocemente nas alterações manifestadas. Os tratamentos para as patologias hematológicas podem ser feitos pela radioterapia, quimioterapia e o transplante de células tronco hematopoéticas (TCTH). Na maioria dos casos, a combinação destas modalidades terapêuticas faz-se necessária para o alcance do controle da doença. Após ser submetido à quimioterapia e/ou radioterapia o paciente é encaminhado para o TCTH almejando-se a cura. Estes tratamentos são altamente agressivos, acarretando prejuízos em todas as dimensões da vida do doente. A compreensão da trajetória da pessoa acometida por uma doença, desde o início dos sinais e sintomas até as perspectivas para o futuro, identificando suas idéias e condutas na luta pela sobrevivência, parte do entendimento do contexto sociocultural em que está inserida. Nessa perspectiva, o objetivo deste estudo foi identificar os modelos explicativos (MEs) para o TCTH alogênico aparentado, na visão de um grupo de pacientes. Para o seu alcance, estabelecemos como base teórica a antropologia médica, o método do estudo de caso qualitativo e a técnica de análise de conteúdo indutivo. Participaram deste estudo 11 pacientes, sendo três com leucemia mielóide aguda, quatro com leucemia mielóide crônica, dois com leucemia linfóide aguda e dois com anemia aplástica grave. As seis mulheres e os cinco homens formam um grupo de adultos em idade produtiva e suas características sociais mostram que são pessoas oriundas da classe social popular. Realizamos entrevistas semi-estruturadas, norteada por questões que integram a construção dos modelos explicativos. Posteriormente compilamos cada uma das entrevistas e seguimos para a análise de dados que se realizou em duas etapas. Reunimos as entrevistas e após várias leituras elencamos as categorias temáticas: da trajetória da doença ao tratamento especializado, os sentidos dados à doença e aos tratamentos, o lidar com a doença e os tratamentos e a vida após os tratamentos. Por estas categorias apreendemos os diversos elementos constitutivos dos modelos explicativos do adoecer e do submeter-se ao TCTH alogênico, onde evidenciamos a influência da cultura em que estão inseridos os participantes. Os entrevistados retrataram os sinais e sintomas da doença, a percepção do estar doente e suas causas, a difícil busca pela assistência à saúde, os tratamentos caseiros, a necessidade das terapêuticas, a existência de um doador compatível, as complicações e suas consequências, as dificuldades financeiras, os cuidados e as mudanças no modo de vida após os tratamentos e as perspectivas de futuro. Concluímos que a cultura é um sistema de referência para as pessoas de um grupo social, que fornece formas de pensar e agir sobre uma determinada situação ou evento. Evidenciamos exemplos da trajetória percorrida pelos participantes em busca da cura, almejando instigar os profissionais da saúde, em especial à equipe de enfermagem, que cuidam destes pacientes a olhá-los em todas as suas dimensões da vida, desejando um cuidado integral e diferenciado, integrando conhecimentos culturais ao modelo biomédico.
People with hematologic disorders face physical, emotional and social alterations. Thus, acting in this area means to be willing to care for the individual, precociously perceiving and intervening in the revealed changes. Hematologic pathologies can be treated by radiotherapy, chemotherapy and hematopoietic stem cell transplantation (HSCT). In most cases, the combination of these types of therapy is needed to control the disease. After undergoing chemotherapy and/or radiotherapy the patient is subject to HSCT, aiming the cure. These treatments are highly aggressive, causing damages in all dimensions of patients lives. Understanding patients sociocultural context is necessary to comprehend the trajectory of someone affected by a disease, since the beginning of the signs and symptoms up to the perspectives for the future, identifying their ideas and behaviors in the fight for survival. In this way, this study aimed to identify the explanatory models (EMs) for allogeneic HSCT of related donors, in the view of a group of patients. In order to achieve this, the medical anthropology, the qualitative case study method and the inductive content analysis technique were established as theoretical bases. Eleven patients participated in the study, three with acute myeloid leukemia, four with chronic myeloid leukemia, two with acute lymphoid leukemia and two with severe aplastic anemia. The six women and five men are adults at reproductive age and their social characteristics show they are from the lower social class. Semi-structured interviews guided by questions that are part of the construction of the explanatory models were carried. Afterwards the interviews were compiled and analyzed in a two-phase data analysis. Interviews were gathered and after several readings, the following thematic categories were listed: from the trajectory of the disease to specialized treatment, the meanings given to the disease and treatments, dealing with the disease and treatments, and life after treatments. Different elements of the explanatory models of becoming ill and undergoing allogeneic HSCT emerged from these categories. The influence of participants cultural context was evidenced. Interviewed subjects reported the signs and symptoms of the disease, the perception of being ill and its causes, the difficult search for health care, home treatments, the need of medications, the existence of a compatible donor, complications and their consequences, the financial difficulties, care and changes in the lifestyle after treatments and the perspectives of future. It is concluded culture is a reference system to people from a social group, which provides ways of thinking and acting about a certain situation or event. Examples of the trajectory followed by the participants in the search for care is evidenced, aiming to motivate health professionals, specially from the nursing team, who delivery care to these patients, to look them in all dimensions of their lives, to provide a comprehensive and differentiated care, integrating cultural knowledge to the biomedical model.

As Síndromes mielodisplásicas (SMD) são doenças clonais da célula progenitora hematopoética, cursando com citopenias, medula óssea displástica e tendência à evolução para leucemia. As SMD secundárias estão associadas a fatores de risco como doenças congênitas (Anemia de Fanconi), doenças hematológicas adquiridas (aplasia medular, HPN), exposição à quimioterápicos (alquilantes, inibidores de topoisomerase II) e radioterapia e substâncias químicas (benzeno, petróleo). Agentes imunossupressores associados ou não a fatores hemopoéticos particularmente utilizados para tratamento da Aplasia medular também se associam à SMD secundária. A OMS recentemente adotou o termo síndrome mielodisplásica/neoplasia mielóde (SMD/NM) relacionada à terapêutica para englobar casos de neoplasias mielóides que preencham critérios morfológicos não somente de SMD, mas também de leucemia mielóide aguda (LMA) ou neoplasias mieloproliferativas. O objetivo do trabalho foi analisar dados clínicos, morfológicos e citogenéticos de 42 portadores de SMD/NM secundária em uma coorte de pacientes diagnosticados no SH-HCFMUSP no período de 1987 a 2008. Vinte e três pacientes (54,8%) eram homens, com mediana de idade de 53,5 (4-88) anos. 45,2% eram portadores de doenças onco-hematológicas, 26,2% de anemia aplástica, 14,3% de tumores sólidos e 14,3% de outras doenças (auto-imunes e transplante de órgãos sólidos). 33% dos pacientes utilizaram exclusivamente QT, 26% combinação QT e RT, 2% RT isolada e 28% agentes imunossupressores. Cinco (11,9%) pacientes haviam sido submetidos a TCTH autólogo para tratamento de doença oncohematológica prévia. A mediana da latência entre a doença primária e a SMD secundária foi de 85 meses (23- 221 meses). Oito pacientes foram submetidos ao TCTH alogênico aparentado para tratamento da SMD secundária. Anemia, neutropenia, plaquetopenia e blastos circulantes foram observados em 64,3%, 54,8%, 78,6% e 26,2% dos casos respectivamente. Cerca de 1/3 dos aspirados medulares apresentavam hemodiluição, 29,7% apresentavam hipocelularidade global, 62,2% apresentavam contagem de blastos superior a 5% e 14,3% sideroblastos em anel acima de 15%. Displasia da série eritróide, granulocítica e megacariocítica foi observada em 79,4%, 77,1% e 68,2% dos casos respectivamente. A histologia medular realizada em 22 casos revelou hipocelularidade global, ALIPs e nódulos linfóides em 9,1%, 23,8% e 40,9% dos casos. A detecção por imunoistoquímica de células CD34>1%, CD117>1%, agrupamento de células CD34+ e de CD117+ e da proteína p53+ foi observada respectivamente em 77,2%, 82,3%, 59%, 29,4% e 33,3% dos casos. Anormalidades clonais foram observadas em 84,3% dos casos, com grande predomínio das não balanceadas (96%), sendo 37% com monossomia 7, 44,4% cariótipos complexos e 18% com outras anormalidades . A mediana de sobrevida de sobrevida global foi de 5,7 meses, pacientes submetidos ao TCTH alogênico para tratamento da SMD/NM secundária tiveram mediana de 40 meses (p=0,007). Fatores associados à pior sobrevida incluíram: doença oncohematológica prévia, baixa contagem plaquetária, elevação de DHL e ferritina, presença de células CD117+ agrupadas, imunoexpressão positiva da p53, citogenética anormal, IPSS intermediário II ou alto risco. Nenhum parâmetro estudado do aspirado medular se associou à sobrevida. Houve tendência à associação da imunoexpressão positiva de p53 a cariótipo anormal e IPSS de maior risco. Não se observou associação entre a presença de ALIP, porcentagem de blastos na morfologia medular e células CD34+ e CD117+. Estes dados reforçam a importância da análise citogenética e da imunoistoquímica da biópsia de medula óssea para diagnóstico e prognóstico das SMD secundárias e do TCTH alogênico no seu tratamento. Mais estudos com maior número de casos devem ser realizados para confirmar a importância do escore IPSS na SMD secundárias, provavelmente substituindo a porcentagem de blastos ao aspirado medular, pela presença de células precursoras detectadas por imunoistoquímica
Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders, characterized by cytopenias, dysplastic bone marrow (BM) and propensity to progress to acute myeloid leukemia. Secondary MDS are associated with risk factors such as congenital disorders (Fanconis anemia), acquired bone marrow failures, exposure to chemotherapy (alkylating agents, topoisomerase II inhibitors) agents and radiation and chemicals (benzene, petroleum). Immunosuppressive agents associated with hematopoietic growth factors are also associated with secondary MDS. The WHO classification has recently adopted the term therapy-related myeloid neoplasms for cases of myeloid malignancies that fulfill morphological criteria not only for MDS but also for AML or myeloproliferative neoplasms.The aim of the study was to analyze clinical, morphological and cytogenetic features of 42 patients with secondary MDS/MN in a cohort of patients diagnosed at our institution from 1987 to 2008. 23 patients (54.8%) were male, median age 53.5 (4-88) years. 45.2% had primary hematologic malignancies, 26.2% aplastic anemia, 14.3% solid tumors and 14.3% other diseases (autoimmune diseases and solid organ transplantation). 33% had undergone chemotherapy alone, 2% RT alone, 26% both modalities and 28% immunosuppressive agents. Five (11.9%) patients had undergone autologous HSCT for treatment of previous malignancies. The median latency between the primary disease and secondary MDS/MN was 85 (23-221) months. Eight patients underwent allogeneic HSCT (allo- HSCT) for treatment of related secondary MDS. Anemia, neutropenia, thrombocytopenia and peripheral blasts were observed in 64.3%, 54.8%, 78.6% and 26.2%, respectively. BM aspirates was poorly representative in 1/3 of cases, 29.7% global hypocellularity, 62.2% more than 5% of blast counts and 14.3% more than 15% of ring sideroblasts. Dysplasia in erythroid, granulocytic and megakaryocytic series was observed in 79.4%, 77.1% and 68.2%, respectively. Twenty two BM biopsies were performed. Global hypocellularity, ALIP and lymphoid nodules were shown in 9.1%, 23.8% and 40.9%. The immunohistochemistry showed more than 1% of CD34+ and CD117+ cells, clusters of CD34+ and CD117+ and immunoexpression of p53 protein in 77.2%, 82.3%, 59%, 29.4% and 33.3%, respectively. Clonal abnormalities were observed in 84.3% of cases with high prevalence of unbalanced (96%) rearrangements. 37% showed monosomy 7 and 44.4% complex karyotypes. The median overall survival was 5.7 for all patients and 40 months for patients treated with allo-HSCT (P=0.007). Hematologic malignancies, low platelet count, serum high LDH and ferritin, detection of CD117+ clusters, positive immunoexpression of p53, abnormal cytogenetics, intermediate-II or high-risk IPSS groups were associated with poor survival. No parameter studied from bone marrow aspirate had impact in survival. p53 expression was associated to abnormal karyotype (P=0.092) and IPSS risk (P=0.054). There was no association between the presence of ALIP, BM blast counts and immunoexpression of CD34+ and CD117+. Our study shows that cytogenetic analysis and BM immunohistochemistry are very important in diagnosis and prognosis, and that allo-HSCT could improve the survival of secondary MDS/MN. More studies with larger numbers of cases should be conducted to confirm the importance of the IPSS for secondary MDS, probably replacing the bone marrow aspirate blast counts by the immunohistochemistry detection of precursor cells

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

As Neoplasias mieloproliferativas (NMP) representam um vasto grupo de doenças clonais hematológicas malignas com três elementos principais: Policitemia vera (PV), Trombocitemia essencial (TE), e Mielofibrose Primária (MFP). JAK2 é uma proteína citoplasmática com atividade de tirosina quinase com função na transdução de várias vias na hematopoiese. A identificação da mutação do gene JAK2 (JAK2V617F) nas PV, TE e MFP representa um importante avanço para a compreensão da biologia destas NMPs. Variações marcantes na frequência desta mutação são observadas entre os diferentes estudos e acredita-se que um dos fatores responsáveis por estas diferenças seja a sensibilidade do método utilizado. Atualmente, diversas técnicas para detecção de JAK2V617F têm sido utilizadas, testadas e validadas quanto à sua sensibilidade e especificidade, entre elas: PCR RFLP (Restriction Fragment Lenght Polymorphysm), ARMS PCR (Amplification-Refractory Mutation System), HRM (High-Resolution Melt Analysis) e Sequenciamento pela técnica de Sanger. Neste estudo foram realizadas todas as metodologias citadas anteriormente para a detecção da mutação de JAK2V617F em amostras de sangue de 136 pacientes (PV=20; MFP=20; TE=28; suspeita de NMP=68). Os resultados obtidos foram concordantes para as quatro técnicas empregadas nos pacientes com PV e MFP, já nos pacientes com TE as metodologias PCR-ARMS e PCR-HRM detectaram a mutação JAK2V617F em 67,8% enquanto o PCR-RFLP e o Sequenciamento pela técnica de Sanger foi 71,4% e 64,2% respectivamente. Nos casos onde houve suspeita diagnóstica de NMP também foram encontradas discordâncias entre as metodologias PCR-RFLP (4,4%) e PCR-HRM (1,5%) quando comparadas ao PCR-ARMS (3%) e o Sequenciamento (3%). O PCR-ARMS foi considerado nesse estudo como a melhor técnica para a detecção da mutação JAK2V617F, devido o menor risco de contaminação cruzada durante a reação, baixo tempo de execução, além da sua capacidade de determinação da carga alélica de JAK2, importante para o acompanhamento do paciente
Myeloproliferative neoplasms (MPN) represent a large group of clonal hematologic malignant diseases with three main members: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Mielofibroses (PMF). JAK2 is a cytoplasmic tyrosine kinase protein and is important in different signal transduction pathways. Identification of JAK2V617F mutation in PV, ET and PMF is an important advance for understanding the biology of MPN. Differences in the frequency of this mutation are reported among different studies and it is believed that technical sensitivity could be the major reason for this variability. Currently, several techniques for detection of JAK2V617F have been developed, tested and validated for their sensitivity and specificity, including: PCR-RFLP (Restriction Fragment Lenght Polymorphysm), PCR-ARMS (Amplification Refractory Mutation System), PCR-HRM (High-Resolution Melt analysis) and Sanger Direct Sequencing. The present study, evaluated all four molecular diagnostic methods mentioned above blood samples from 136 patients (PV=20; MFP=20; ET=28 and other MPN=68). Comparable results were observed for PV and PMF when all technics were applied. Patients with diagnosis of ET JAK2V617F mutations were detected in 67.8% when PCR-ARMS and PCR-HRM were used whilst PCR-RFLP and direct sequencing detected 71.4% and 64.2% respectively. In 68 patients with suspicion of MPN discordant results were seen between PCR-RFLP (4.4%) and PCR-HRM (1.5%) when compared to PCR-ARMS (3%) and direct sequencing (3%) related to JAK2V617F frequency. In conclusion PCR-ARMS was considered the most reliable methodology for JAK2V617F detection by presenting the lowest risk for cross contamination, less laborious, and the ability in determining allele burden that is becoming an important tool for risk stratification

Indiana University-Purdue University Indianapolis (IUPUI)
Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2016
Myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). MPN are characterized by clonal proliferation of myeloid progenitors leading to erythrocytosis, thrombocytosis and/or leukocytosis, and risk of hemorrhagic and thrombotic events, as well as myelofibrosis and blast transformation. The discovery of somatic mutations in MPN, namely JAK2 V617F, JAK2 exon 12, MPL, and CALR mutations, has permitted a more specific approach to diagnosis and treatment. The prevalence of JAK2 V617F mutations is higher than 95% in PV, 50-75% in ET and 40-75% in PMF. JAK2 exon 12 mutations are specific of PV. 20-30% of ET and PMF patients present a CALR mutation. The screening of mutations strengthens the diagnosis of MPN since 97% of MPN have at least one somatic mutation. Interestingly, different mutations grant different phenotype and prognosis. Of particular importance, CALR mutations grant a favorable prognosis in ET and PMF, while ASXL1 mutations confer a poorer outcome. In fact, the use of CALR/ASXL1 status for the prognostication of patients has increased clinical value and was suggested for guidance of therapy in PMF. The increasing importance of mutations in the management of MPN warrants a revision of current diagnostic criteria and prognostic models.
As neoplasias mieloproliferativas (MPN) policitémia vera (PV), trombocitémia essencial (ET) e mielofibrose primária (PMF) são caracterizadas por proliferação clonal de progenitores mielóides, levando a eritrocitose, trombocitose e/ou leucocitose, risco de eventos hemorrágicos e trombóticos, mielofibrose e transformação blástica. A descoberta de mutações somáticas, nomeadamente JAK2 V617F, JAK2 exão 12, MPL e CALR, tem permitido um diagnóstico e tratamento mais específico. A prevalência da JAK2 V617F na PV ultrapassa os 95%, enquanto na ET é de 50-75% e na PMF de 40-75%. As mutações JAK2 exão 12 são específicas da PV. 20-30% dos doentes com ET ou PMF apresentam mutação na CALR. A identificação de mutações reforça o diagnóstico pois 97% das MPN apresentam pelo menos uma mutação. É interessante notar que diferentes mutações conferem diferentes fenótipos e prognósticos. De particular relevo, as mutações da CALR conferem bom prognóstico na ET e na PMF, ao passo que mutações da ASXL1 atribuem piores desfechos. De facto, a utilização do estado da CALR/ASXL1 para a definição do prognóstico na PMF tem valor clínico e foi sugerida com ferramenta para guiar a terapêutica. A importância crescente das mutações na gestão das MPN implica uma revisão dos atuais critérios de diagnóstico e modelos de prognóstico.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Haematology. Johannesburg, March 2015
The incidence of leukaemia in South Africa is 2.5 per 100 000 and has increased due to HIV. Accurate and timeous diagnosis of leukaemia directly impacts success of patient treatment and consequent survival. Usually the Full Blood count (FBC), white blood cell (WBC) differential count and review of the peripheral blood smear alerts the clinician to the possibility of leukaemia. However the number of qualified and skilled technologists in peripheral and central laboratories is on a continual decline making the performance of the critical function of peripheral blood review a challenge. The Advia 2120 haematology analyser performs a WBC and differential count using principles of flow cytometry and the cytograms generated can be used to classify haematological malignancies through the Peroxidase and nuclear density analysis (PANDA) classification system. The presence of myeloperoxidase (MPO) activity in 3% or more leukaemic blasts confirms acute myeloid leukaemia, and enzyme activity can be detected by immunophenotypic analysis or conventional cytochemistry . Research on the comparison of the Advia 2120 and manual morphologic assessment in the classification of leukaemias is limited in the South African setting, where leukaemia often coincides with infection. The aim of this study was to determine if the FBC, differential count and cytogram assessment by the Advia 2120 using the PANDA classification is as reliable as morphologic assessment in the initial classification of haematological malignancies from peripheral blood samples when using flow cytometry as the gold standard.. 150 cases of confirmed leukaemia were collected. The diagnosis obtained from either PANDA analysis and/or morphological assessment was compared to the diagnosis obtained by immunophenotypic analysis. Secondly, the MPO activity obtained by the Advia peroxidase cytogram was compared to the MPO obtained by conventional methods of immunophenotypic analysis and/or cytochemistry. Using the PANDA analysis system, only 48% (72/150) of cases overall were accurately classified. The inaccuracy was 9.3% (14/150) and 42.7% of cases could not be classified. The positive predictive value (PPV) was 88%. The most significant finding was all of the acute Page | iv promyelocytic leukaemia (APL) cases (8/8) had a distinct pattern and were accurately classified on cytogram analysis alone. Accurate sub-classification of other types of acute myeloid leukaemia using PANDA analysis alone was inconsistent. However, the accuracy in classifying leukaemia was improved when the Advia cytogram was used in conjunction with morphological analysis, as 90% (135/150) of cases were accurately classified. The sensitivity and specificity of the peroxidase cytogram in evaluating myeloperoxidase (MPO) activity was 85% and 88.6% respectively. The agreement between cytogram peroxidase activity and the reference methods was 89.1% and the Cohen’s kappa was 76.9%. To the best of our knowledge, there is no data comparing peroxidase activity on the cytogram to other methods. In conclusion, it was shown that the routine use of the Advia cytograms in conjunction with the morphology findings provides invaluable information in the initial screening of leukaemia. In cases with indistinct morphology, the cytograms have the potential to aid in a provisional classification. The peroxidase activity from the cytogram could be used as a surrogate marker for myeloperoxidase activity in leukaemia. Moreover, a tentative diagnosis of an APL is possible by simple analysis of the cytogram resulting in earlier diagnosis which could be life-saving.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
Nas últimas décadas, tem sido cada vez mais reportado na literatura a presença de doenças múltiplas num mesmo indivíduo, nomeadamente a coexistência de mais do que uma Doença Linfoproliferativa Crónica de células B (DLPC-B), a presença simultânea de uma Gamapatia Monoclonal e/ou Mieloma Múltiplo com Síndrome Mielodisplásica (SMD) e, mais recentemente casos diagnosticados com DLPC-B concomitante a SMD. Contudo, relativamente às últimas duas doenças, não se sabe ao certo qual o impacto da presença simultânea destas duas entidades na sobrevida global do doente, nem de que modo poderá influenciar o valor prognóstico nestes casos.Neste trabalho, avaliou-se a capacidade de um painel de diagnóstico fenotípico construído de forma a identificar e caracterizar fenotipicamente as células B patológicas numa amostra, de forma a poder classificar a doença de acordo com a classificação da OMS, e conseguir concomitantemente identificar rasgos fenotípicos displásicos nas linhas a neutrófilo (avaliação da expressão de CD10, CD43, CD31, CD305, CD11c e CD95), monocítica (CD43, CD31, CD305, CD11c, CD81, CD95, CD49d, CD39 e HLA-DR); e eritroide (CD43, CD95 e CD49d).Foram avaliadas 75 amostras de aspirados medulares de indivíduos, cuja idade variava entre 47 e 93 anos (74,5 ± 10,1 anos), sendo 48 do sexo masculino e 27 do sexo feminino; referentes a 51 amostras de doentes com diagnóstico de DLPC-B, 10 amostras de linfocitose B monoclonal (MBL), 8 amostras de doentes com diagnóstico de SMD e 6 amostras de MO normais. O grupo DLPC-B foi dividido em doentes diagnosticados com Linfomas não Hodgkin-B e em casos diagnosticados com Leucemia Linfocítica Crónica de células B/ Linfoma Linfocítico. Posteriormente, estes foram divididos em casos com displasia morfológica > a 10% das células pertencentes a uma das linhas hematopoiéticas; casos com displasia morfológica < a 10%; casos sem qualquer evidência de displasia morfológica. Em todas estas amostras procedeu-se à caracterização fenotípica das células B, por citometria de fluxo, com base no painel de diagnóstico preconizado pelo consórcio EuroFlow. Numa primeira abordagem comparou-se a expressão dos receptores acima mencionados entre o grupo SMD e Controlo, tendo-se observado diferenças estatisticamente significativas para CD10, CD305 e CD95 na linha a neutrófilo; para CD39 e HLA-DR na linha monocítica e para CD43, CD49d e CD95 na linha eritroide. Fomos depois avaliar se a expressão destes receptores se encontrava alterada nos diferentes grupos de DLPC-B/MBL gerados, tendo-se observado diferenças estatisticamente significativas no grupo com mais de 10% de displasia, para CD10 na linha a neutrófilo, para CD39 e HLA-DR na linha monocítica e para CD43 na linha eritroide. Os resultados obtidos apontam para a utilidade do painel de diagnóstico de DLPC-B na identificação de rasgos fenotípicos displásicos nas linhas hematopoiéticas estudadas, permitindo suspeitar da presença de SMD concomitante com uma DLPC-B. Este estudo permitiu, também, identificar novos receptores expressos nas diferentes linhas estudadas, ainda não descritos na literatura como sendo de interesse para o diagnóstico de SMD.
In the last decades, the presence of multiple diseases in the same individual, such as the coexistence of more than one B-cell Chronic Lymphoproliferative Disorder (B-CLPD), the simultaneous presence of Monoclonal Gamapathy and/or Multiple Myeloma with Myelodysplastic Syndrome (MDS), and more recently cases diagnosed with B-CLPD concomitant with MDS. However, for the latter two diseases, the impact of the simultaneous presence of these two entities on overall patient survival is not known, nether how the prognostic value can be influenced in these cases. In this work, the ability of a panel of phenotypic diagnosis constructed to identify and characterize phenotypically the pathological B cells in a sample, in order to be able to classify the disease according to WHO classification, concomitantly to identify phenotypic dysplastic features in the neutrophil (evaluation of the expression CD10, CD43, CD31, CD305, CD11c and CD95), monocytic (CD43, CD31, CD31, CD11c, CD81, CD95, CD49d, CD39 and HLA-DR) and erythroid lineage (CD43, CD95 and CD49d), Seventy-five samples of spinal aspirates were evaluated, ranging in age from 47 to 93 years (74.5 ± 10.1 years), of which 48 were male and 27 were female; 51 samples from patients diagnosed with B-CLPD, 10 samples from monoclonal B-cell lymphocytosis (MBL), 8 samples from patients diagnosed with MDS and 6 samples from normal bone marrow (BM). The B-CLPD group was divided into patients diagnosed with B-cell Non-Hodgkin's Lymphoma and in cases diagnosed with B-cell Chronic Lymphocytic Leukemia / Lymphocytic Lymphoma. Subsequently, these were divided into cases with morphological dysplasia >10% of the cells belonging to one of the hematopoietic lineages; Cases with morphological dysplasia <10%; Cases without any evidence of morphological dysplasia. In all these samples the phenotypic characterization of B cells was performed by flow cytometry, based on the diagnostic panel recommended by the EuroFlow consortium. In a first approach the expression of the above mentioned receptors was compared between the MDS and Control groups, with statistically significant differences being observed for CD10, CD305 and CD95 in the neutrophil lineage; For CD39 and HLA-DR in the monocytic lineage, and for CD43, CD49d and CD95 in the erythroid lineage. Then, we evaluated whether the expression of these receptors was altered in the different B-CLPD/ MBL groups, and statistically significant differences were observed in the group with more than 10% dysplasia, for CD10 in the neutrophil lineage, for CD39 and HLA -DR in the monocytic lineage and for CD43 in the erythroid lineage. The results obtained point to the usefulness of the B-CLPD diagnostic panel in the identification of dysplastic phenotypic features in the hematopoietic lineages studied, allowing to suspect the presence of concomitant MDS with a B-CLPD. This study also allowed to identify new receptors expressed in the different lineages studied, not yet described in the literature as being of interest for the diagnosis of MDS.