published_or_final_version / Pathology / Master / Master of Philosophy

Thesis (M. Phil.)--Universiry of Hong Kong, 1994. / Includes bibliographical references (leaves 111-121).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 76-82).

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Understanding which cells within a cancer are responsible for its initiation and propagation is vital if we are to achieve cure. If cancer stem cells are the only population able to sustain a tumour long term, designing therapeutic strategies to target this population will give medical science the best chance of long-term cure. Significant controversy remains over the existence of cancer stem cells, predominantly due to the lack of a sensitive human cancer stem cell assay. This thesis investigates whether two haematological malignancies, myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML) can only be driven by rare and distinct cancer stem cells. We have demonstrated that low and intermediate-1 risk MDS is driven solely by the stem cell (Lin- CD34+ CD38- CD90+ CD45RA-) by developing a novel genetic approach, tracing all somatic mutations and karyotypic abnormalities back to this population. Prior to this study, very little was known about the clonal architecture of CMML. By performing detailed phenotypic, functional, molecular and genetic analysis of patients with CMML, we were able to demonstrate that the most likely candidate driver cell in these patients was also the stem cell rather than any of the down-stream progenitors. Currently, effective therapeutic strategies for MDS or CMML are very limited. Allogeneic stem cell transplantation is the only potential cure and not suitable for most patients. Cancer stem cells, including MDS stem cells are known to be highly quiescent and selectively resistant to therapy. Having demonstrated that both MDS and CMML were driven by stem cells, we developed a novel therapeutic targeting strategy. Using the thrombopoietin receptor agonist, Romiplostim, we were able to activate stem cells and enhance their subsequent sensitivity to chemotherapy dramatically. This approach may facilitate improved remission rates and prevent cancer stem cell driven relapse in many diseases.

A SÃndrome MielodisplÃsica à uma hemopatia clonal de alta prevalÃncia em idosos Anemia à uma caracterÃstica marcante dessa doenÃa Pacientes com dependÃncia de suporte transfusional tem pior prognÃstico Depois das complicaÃÃes relacionadas à prÃpria doenÃa as complicaÃÃes cardiovasculares sÃo a principal causa de morte Novas tÃcnicas ecocardiogrÃficas como o Doppler tecidual e speckle-tracking podem ser Ãteis na anÃlise da funÃÃo cardÃaca nesse grupo Neste estudo foi avaliado um grupo de 34 pacientes e 14 controles saudÃveis emparelhados por sexo e idade sendo submetido à ecocardiograma convencional Doppler tecidual e avaliaÃÃo da deformaÃÃo miocÃrdica (strain) Os pacientes foram subdivididos entre dependentes (13) ou nÃo de suporte transfusional (21) e comparados aos controles Dentre os 13 pacientes do grupo dependentes havia 10 com sobrecarga de ferro (ferritina >1.000 ng/mL) Os pacientes dependentes de suporte transfusional apresentaram maiores volumes diastÃlico e sistÃlico do ventrÃculo esquerdo em relaÃÃo aos controles (p = 0,047 e 0,039) O volume do Ãtrio esquerdo indexado foi maior no grupo de dependentes em relaÃÃo ao grupo controle (p = 0,003) A funÃÃo diastÃlica do ventrÃculo esquerdo (VE) por Doppler convencional e tecidual (razÃo E/A e razÃo E/Eâ) foi normal no grupo de pacientes e nÃo apresentou diferenÃa significante entre os grupos (p = 0,15 e 0,90) Na avaliaÃÃo da funÃÃo sistÃlica do VE por fraÃÃo de ejeÃÃo e por deformaÃÃo miocÃrdica (strain longitudinal global) nÃo houve desvio da normalidade nem diferenÃas entre os grupos (p = 0,71 e 0,097) A espessura do septo interventricular foi maior nos pacientes com ferritina > 1.000 ng/mL (p = 0,012) O nÃvel de hemoglobina mas nÃo o de ferritina apresentou correlaÃÃo com os volumes esquerdos (Ãtrio: r = -0,53 e p = 0,013 / ventrÃculo: r = -0,4 e p = 0,019) Nossa amostra nÃo apresentou disfunÃÃo global nem sistÃlica nem diastÃlica mesmo à anÃlise por novas tÃcnicas de ecocardiograma como Doppler tecidual e deformaÃÃo miocÃrdica (strain) O nÃvel de hemoglobina menor que 8 g/dL foi marcador precoce de pior funÃÃo ventricular nos nossos pacientes com SÃndrome MielodisplÃsica / Myelodysplastic syndrome is a clonal disorder of hematopoietic tissue highly prevalent on elderly Anemia is one of most striking feature of this disorder Patients with transfusional dependence have a poor prognosis Following complications related to the own illness cardiovascular complications are the leading cause of death New echocardiographic techniques such as Tissue Doppler and speckle-tracking may be useful on assessment of the myocardial function in these patients A group with 34 patients and 14 healthy controls matched by sex and age was subjected to conventional echocardiography Tissue Doppler and assessment of myocardial deformation (strain) Patients were divided between those with (13) or without (21) transfusional dependence and compared to controls In the group of transfusional dependence there were 10 subjects with iron overload (serum ferritin levels > 1.000 ng/mL) Those with transfusion dependence had bigger left systolic and diastolic ventricular volumes than controls (p = 0,047 and 0,039) The indexed left atrium volume was larger on those with transfusion dependence compared to controls (p = 0,003) The left ventricular diastolic function assessed by tissue and conventional Doppler (E/A and E/Eâ ratios) was normal in the patient group and has not difference between them (p = 0,15 and 0,9) On the assessment of the systolic left ventricular function by ejection fraction and myocardial deformation (global longitudinal strain) there was no difference between groups or from reference values (p = 0,71 and 0,097) The interventricular septum thickness was larger in the group with serum ferritin > 1.000 ng/mL than patients with ferritin < 1.000 ng/mL (p = 0,012) The hemoglobin level but not ferritin showed linear correlation with the left volumes (atrium: r = -0,53 with p =0,013 / ventricle: r = -0,4 with p = 0,019) Our sample doesnât show diastolic nor systolic global dysfunctions yet with new techniques of tissue Doppler and myocardial deformation (strain). Hemoglobin below 8 g/dL was an early marker of worst ventricular function in our patients with myelodysplastic syndrome

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

No description available.

Les syndromes myélodysplasiques (SMD) peuvent être conçus comme des conditions pré-leucémiques dans lesquelles l’apoptose avorte les produits de différenciation de cellules souches mutées, potentiellement malignes. Néanmoins, peut-être à cause d’une inhibition progressive de l’apoptose, les SMD se transforment fréquemment en leucémies aiguës myéloïdes (LAM). Nos données indiquent que les SMD à faible risque se caractérisent par l’absence d’activation de NF-κB au sein des cellules portant des altérations cytogénétiques typiques. Par contre, dans les SMD à haut risque de transformation en LAM (ainsi que dans les LAM post-SMD), les cellules souches hématopoïétiques et leurs produits de différenciation montrent une translocation activatrice des sous-unités p50/p65 de NF-κB. L’utilisation d’antagonistes de IKK provoque une inhibition de NF-κB conduisant à une apoptose accélérée, ainsi l’activation de NF-κB serait responsable de la suppression progressive de l’apoptose et donc de la transformation maligne. Ce projet de thèse a consisté à comprendre les mécanismes impliqués dans la dérégulation de l’apoptose dans les SMD/LAM ; ainsi qu’à utiliser des technologies de criblage pour permettre une meilleure compréhension des voies de signalisation impliquées, et à adapter de nouveaux outils d’analyse. Au cours d’une première étude, nous avons montré que les inhibiteurs de méthyltransférase de l’ADN et les inhibiteurs d’histones déacétylases induisent efficacement l’apoptose dans la lignée cellulaire SMD/LAM P39, parallèlement à une inhibition de la translocation de NF-κB du cytoplasme au noyau. Dans une seconde étude, nous avons montré que l’inhibition pharmacologique du récepteur Flt3 induit une inhibition de la voie NF-κB, et pourrait être une cible thérapeutique pertinente. Dans une troisième étude, nous avons montré que l’auto-activation d’ATM chez les patients atteints de SMD/LAM joue un rôle dans l’activation constitutive de NF-κB suggérant qu’ATM serait également une bonne cible thérapeutique dont l’inhibition pourrait réduire le défaut d’apoptose des cellules SMD et LAM. Et enfin, grâce à l’optimisation d’une technique d’analyse d’images à haut débit, nous avons identifié deux composés capables d’induire la mort cellulaire des lignées cellulaires LAM in vitro : le zinc pyrithione et la ouabain. Leurs effets d’inhibition du signal de survie NF-κB, conduisant à une réduction de l’expression de protéines anti-apoptotiques, suggèrent que ces composés pharmaceutiques pourraient être utilisés comme des agents anti-leucémiques. Ce projet de thèse nous a permis de mettre en évidence le potentiel anti-leucémique de différents agents impliqués dans les principales voies de signalisation de l’apoptose dérégulées dans les SMD/LAM, qui pourraient prochainement servir de cibles pour de nouveaux essais thérapeutiques. / Myelodysplastic syndrome (MDS) is a group of hematopoietic stem cell disorders that is characterized by an ineffective hematopoiesis (finaly leading to blood cytopenias) and by a high risk of progression to acute myeloid leukemia (AML). It can therefore be viewed as a preleukemic condition in which apoptosis aborts the differentiation products of potentially malignant mutated (stem) cells. The progression of MDS into AML is associated with progressive inhibition of apoptotsis (by e.g. the expression of antiapoptotic proteins) and a negative prognostic value, suggesting that loss of the apoptotic program could favor the MDS-to-AML transition. Therefore the present project aimed at understanding the mechanisms involved in the deregulation of apoptosis in MDS and AML and the characterization of their underlying signaling pathways by means of standard biochemical and high throughput screening approaches. Our previous work showed that inhibitors of DNA methyltransferases and histone deacetylases effectively induced apoptosis in AML cells in vivo which was associated with an inhibition of NF-κB-dependent transactivation of survival signals. We further found that the pharmacological inhibition of the Flt3 receptor in AML cells decreased NF-κB activation and might therefore constitute a relevant therapeutic target for the treatment of AML. In line with these findings we demonstrated that the constitutive activation of ATM in high-risk MDS and AML patients accounts for the activation of NF-κB suggesting ATM as yet another drugable target for antileukemic therapy. Finally we generated a high throughput image based screening platform, which enabled us to perform large scale drug screening approaches and to identify two compounds with antileukemic properties. Both agent, pyrithione zinc (PZ) and Ouabain (OUA) efficiently induced cell death in AML cells in vitro associated with the inhibition of NF-κB. PZ and OUA exerted significant anticancer effects in vivo, on human AML cells xenografts as well as ex vivo, on CD34+ (but not CD34-) malignant myeloblasts from AML patients. Summarizing this project allowed us to shed some light on the importance of NF-κB during MDS to AML progression and at the same time it helped to identify drugable targets and agents with potential anticancer properties for the treatment of leukemia.

As síndromes mielodisplásicas (SMD) constituem um grupo heterogêneo de doenças hematológicas de origem clonal, caracterizado por hematopoese ineficaz, citopenia e risco de evolução para leucemia mieloide aguda (LMA). As anormalidades citogenéticas adquiridas são marcadores prognósticos bem estabelecidos em SMD. No entanto, a técnica de citogenética metafásica apresenta limitações, incluindo baixa resolução e necessidade de divisão celular, sendo que defeitos cromossômicos podem não ser detectados. Tecnologias baseadas em microarranjo (array) de DNA, como o Single Nucleotide Polymorphism Array (SNP-A), são importantes para avaliação do genoma normal e neoplásico. O SNP-A foi desenvolvido para o estudo de todo o genoma, apresenta uma resolução superior a citogenética metafásica convencional, pode ser realizado em células na interfase, e detecta alterações cromossômicas não visualizadas pela citogenética metafásica. Além disso, o SNPA fornece dados de genotipagem para detecção de perda neutra de heterozigose, também denominada de dissomia uniparental somática. Regiões cromossômicas com deleção, perda neutra de heterozigose ou ganho são comuns em pacientes com neoplasias hematológicas e sugeriu genes candidatos a supressores de tumor e oncogenes. O objetivo do presente estudo foi a caracterização da coorte de pacientes com suspeita clínica de SMD e o uso integrado do método de citogenética convencional e SNP-A no serviço de hematologia da nossa instituição na investigação de alterações citogenéticas em pacientes com SMD e doenças relacionadas. Durante o período do estudo, foram recebidas um total de 114 amostras de pacientes com suspeita clínica de SMD. A análise clínica, morfológica e citogenética permitiu confirmar o diagnóstico de SMD ou doenças relacionadas em 43 pacientes (SMD [n=34], SMD/NMP [n=5], LMA com alterações mielodisplásicas [n=4]). Vinte e um pacientes foram classificados como citopenia idiopática de significado indeterminado (CISI) e 50 indivíduos apresentaram outros diagnósticos. SNP-A foi realizado em 17 pacientes com SMD e doenças relacionadas. Dentre os pacientes selecionados para o SNP-A, anormalidades cromossômicas foram observadas em 6/17 (35%) casos pelo cariótipo convencional e em 8/17 (47%) casos pela técnica de SNP-A. SNP-A não detectou quatro alterações cromossômicas previamente identificadas pela citogenética convencional: duas translocações balanceadas e duas alterações numéricas. SNP-A confirmou os demais achados identificados pela citogenética convencional e detectou um total de 32 novas lesões (1 ganho, 19 perdas e 12 UPDs) em 6 pacientes com SMD ou doenças relacionadas. SNP-A pode complementar a citogenética convencional na detecção de anormalidades cromossômicas em neoplasias mieloides. / Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic diseases, characterized by inefficient hematopoiesis, peripheral blood cytopenias and a risk to progress to acute myeloid leukemia (AML). Acquired chromosomal abnormalities have prognostic value in MDS. However, metaphase cytogenetics has some limitations including low resolution and the requirement of cell division, and chromosomal abnormalities may not be detected. New technologies based on array, the Single Nucleotide Polymorphism Array (SNP-A), are able to evaluate the whole genome. The SNP-A has superior resolution compared to metaphase cytogenetics, may be used in interphase cells, and may detect chromosomal abnormalities not detected by metaphase cytogenetics. In addition, the SNP-A read-out includes genotyping calls and hybridization signal strength, corresponding to gene copy number, allowing detecting copy neutral loss of heterozigosity (CN-LOH), also known as uniparental dissomy (UPD). Deletions, copy neutral loss of heterozigosity or gain are frequent in patients with haematopoietic neoplasms and has already suggested the location of tumor suppressor genes and oncogenes. The aim of this study was to characterize the cohort of patients with clinical suspicion of MDS and to establish the integrative use of the conventional cytogenetic and the SNP-A in the investigation of chromosomal abnormalities in patients with MDS and related diseases followed at our institution. The clinical, morphological and cytogenetic evaluation allowed us to confirm the diagnosis of MDS or related disease in 43 patients (MDS [n=34], MDS/MPN [n=5], AML with myelodysplastic changes [n=4]). Twenty-one patients were diagnosed with idiopathic cytopenia with undetermined significance (ICUS) and 50 patients had other diagnosis. SNP-A were performed in 17 patients with MDS and related disease. Chromosomal abnormalities were observed in 6/17 (35%) cases by metaphase cytogenetics, and in 8/17 (47%) of the cases by SNP-A. SNP-A did not detected two balanced translocations and two numerical alterations previously observed by metaphase cytogenetics. SNP-A confirmed all the other findings observed by metaphase cytogenetics and SNP-A detected a total of 32 new lesions (1 gain, 19 losses and 12 UPDs) in 6 MDS and related diseases. SNP-A may complement metaphase cytogenetics to improve the detection of chromosomal abnormalities in myeloid neoplasms.