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No description available.

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

Acute Myeloid leukemia (AML) is an aggressive haematological malignancy that mainly affects the elderly. Relapse is common and is thought to be due to the presence of chemotherapy resistant leukemic stem cells (LSC). Within the CD34+ disease (>5% of the blast cells expressing CD34) , two subtypes have been identified; an LMPP/GMPlike expanded type and a MPP/CMP-like expanded type, the former is the most common, accounting for around 80% of CD34+ AML. Both the GMP-like and LMPPlike expanded populations show LSC activity. To improve our understanding of the disease and gain better insight in to how to develop treatments, the molecular basis of the disease needs to be investigated. I investigated miRNAs in the GMP/LMPP-like expanded AML. miRNAs are small non-coding RNAs involved in the regulation of mRNA. In recent years miRNAs have been shown to be implicated in many different diseases. To investigate the role miRNAs play in AML, miRNA expression was profiled in leukemic and normal bone marrow. Bioinformatic analysis was then used to examine the different miRNA expression profiles between normal and leukemic marrow. Our study showed that miRNAs are dysregulated in AML. miRNAs from the miR-17-92 and its paralogous cluster miR-106b-92 were amongst the miRNAs to be found down regulated in AML As had been seen previously at an mRNA level, on an miRNA level the LSC populations more closely resembled more mature progenitor populations than HSC and MPP populations, however the LSC populations did display an aberrant stem cell-like miRNA signature.

On note un taux élevé de résistance aux traitements dans la leucémie myéloïde aiguë (LMA). Cette résistance peut être associée aux altérations de TP53. Les « ubiquitin specific peptidases » (USP) sont impliquées dans plusieurs cancers mais leurs rôles ne sont pas élucidés dans les LMA. L’analyse de l’expression génique par RT-PCR quantitative de 21 USP et des gènes de l’axe USP7-MDM2-TP53-CDKN1A dans 111 échantillons de LMA a montré une dérégulation de USP44, USP1, USP28 et CDKN1A dans respectivement 72%, 44%, 25% et 42% des cas. CDKN1A, une cible importante de TP53, pourrait avoir un rôle dans la résistance au traitement. Nous avons développé un modèle expérimental pour évaluer la réponse des cellules leucémiques à la doxorubicine et au nutlin 3, un modulateur non génotoxique de TP53, selon l’expression initiale de CDKN1A. Ce travail préliminaire suggère que certains membres de la famille des USP et CDKN1A pourraient représenter de nouvelles cibles thérapeutiques dans les LMA. / There is a high rate of drug resistance in acute myeloid leukemia (AML) which may be associated with TP53 alterations. The « Ubiquitin specific peptidases » (USP) are involved in several cancers but their roles in AML are not elucidated. Gene expression analysis of 21 USP and genes of the USP7-MDM2-TP53-CDKN1A axis by quantitative RT-PCR in 111 AML samples, showed a deregulation of USP44, USP1, USP28 and CDKN1A in respectively 72%, 44%, 25% and 42% of cases. CDKN1A, an important TP53 target, may have a role in treatment resistance. We have developed an experimental model to assess the response of leukemic cells to doxorubicin and nutlin 3, a non genotoxic TP53 modulator, in relation to the CDKN1A expression level. This preliminary work suggests that some members of the USP family and CDKN1A could represent novel therapeutic targets in AML.

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Student Number : 9807768F - MSc (Med) research report - Faculty of Health Sciences / Fanconi anaemia (FA) is an autosomal recessive disorder characterised by aplastic anaemia (AA) and a high risk of developing acute myeloid leukaemia (AML). It is unknown whether heterozygote carriers are also predisposed to developing these disorders. The black South African population group is ideal for FA mutation screening because the presence of a founder mutation, FANCG 637-643, makes screening relatively straight forward. Three individuals with AML (115 screened) and one with AA (78 screened) were found to be heterozygous for the black South African founder mutation. From our data it seems unlikely that this mutation places heterozygous carriers of the mutation at high risk of developing AML or AA. Three children with AA out of 26 screened, were homozygous for the mutation. This finding reiterates the importance of screening all children with AA for FA. The frequency of certain congenital abnormalities in black South African FA patients was compared to patients described by other research groups. The frequencies of the abnormalities were similar to other FANCG cohorts described but significant differences to a group of FA patients from unspecified complementation groups were found. This difference could be because different complementation groups are associated more or less strongly with specific abnormalities. It was found previously that particular congenital abnormalities in FA patients are associated with a poor haematological outcome. We concluded that black South African FANCG patients have a high risk of early development of AA even though they do not have a high frequency of congenital abnormalities.

A técnica de análise de sobrevivência tem sido muito utilizada por pesquisadores na área de saúde. Neste trabalho foi usada uma distribuição em análise de sobrevivência recentemente estudada, chamada distribuição exponencial generalizada. Esta distribuição foi estudada sob todos os aspectos: para dados completos e censurados, sob a presençaa de covariáveis e considerando sua extensão para um modelo multivariado derivado de uma função cópula. Para exemplificação desta nova distribuição, foram utilizados dados reais de câncer (leucemia mielóide aguda e câncer gástrico) que possuem a presença de censuras e covariáveis. Os dados referentes ao câncer gástrico tem a particularidade de apresentar dois tempos de sobrevida, um relativo ao tempo global de sobrevida e o outro relativo ao tempo de sobrevida livre do evento, que foi utilizado para a aplicação do modelo multivariado. Foi realizada uma comparação com outras distribuições já utilizadas em análise de sobrevivência, como a distribuiçãoo Weibull e a Gama. Para a análise bayesiana adotamos diferentes distribuições a priori para os parâmetros. Foi utilizado, nas aplicações, métodos de simulação de MCMC (Monte Carlo em Cadeias de Markov) e o software Winbugs. / Survival analysis methods has been extensively used by health researchers. In this work it was proposed the use a survival analysis model recently studied, denoted as generalized exponential distribution. This distribution was studied in all respects: for complete data and censored, in the presence of covariates and considering its extension to a multivariate model derived from a copula function. To exemplify the use of these models, it was considered real cancer lifetime data (acute myeloid leukemia and gastric cancer) in presence of censored data and covariates. The assumed cancer gastric lifetime data has two survival responses, one related to the total lifetime of the patient and another one related to the time free of the disease, that is, multivariate data associated to each patient. In these applications there was considered a comparative study with standard existing lifetime distributions, as Weibull and gamma distributions.For a Bayesian analysis we assumed different prior distributions for the parameters of the model. For the simulation of samples of the joint posterior distribution of interest, we used standard MCMC (Markov Chain Monte Carlo) methods and the software Winbugs.

Les cellules Natural Killer (NK) sont jouent un rôle essentiel dans la surveillance des hémopathies malignes. Cependant, les cellules tumorales développent des mécanismes immunosuppresseurs pour échapper à l'immunité cellulaire NK. Ainsi, le maintien ou l'amélioration des performances des cellules NK sont considérés comme des défis majeurs. Les objectifs de cette partie étaient d'évaluer l'impact de la perfusion de cellules NK activées sur la récupération et la biologie des cellules NK circulantes après l'allo-SCT. Des doses croissantes de cellules NK activées par IL-2 ex-vivo ont été perfusées chez des patients atteints de tumeurs malignes hématologiques 3 mois après allo-SCT. Nos résultats ont montré une fréquence plus élevée des cellules NK dans la périphérie des patients traités. Bien que le phénotype immature soit remarquable peu après le traitement, les cellules NK circulantes, présentaient un état d'activation avec un profil de maturation amélioré après 6 mois de traitement. Nous avons également constaté que l'expression des récepteurs NK activateurs (NKG2D, NKp30 et NKp46) augmentait sur les cellules NK circulantes des patients. De plus, ces cellules ont montré une augmentation significative de la capacité de dégranulation ainsi que de la sécrétion de cytokines (IFN-Ƴ et TNF-α) tout au long de l'étude. Ces différences ont notamment été observées chez les patients ayant reçu des doses plus importantes de cellules NK activées. En conclusion, nous supposons que la perfusion de fortes doses de cellules NK activées ex-vivo pourrait être associée à l'amélioration du phénotype et des fonctions des cellules NK au cours de la reconstitution immunitaire après allo-SCT. / Natural killer (NK) cells are effector lymphocytes of the innate immune system that have the ability to kill transformed cells. They play a critical role in hematological malignancies surveillance, however, tumor cells develop immunosuppressive mechanisms to escape NK cell-mediated killing. So, maintaining or improving NK cell performance is considered a major challenge. Our goals are to evaluate the impact of activated NK cells infusion on the recovery and biology of circulating NK cells post allo-SCT.Three different doses (dose 1: 106 NK cell/Kg, n=3; dose 2: 5x106 NK cell/Kg, n=7; dose 3: >5x106 NK cell/Kg, n=6) of ex-vivo IL-2 activated NK cells were infused into patients with hematological malignancies after all-SCT. Our results showed higher frequency of NK cells in the periphery of patients treated with larger doses of activated NK cells. Although the notable immature phenotype shortly after treatment, the circulating NK cells in patients receiving larger doses of activated NK cells displayed more activation status with improved maturation profile after 6 months of treatment. We also found that the expression of activating NK receptors (NKG2D, NKp30, and NKp46) augmented on circulating CD56dim NK cells of patients receiving larger doses of activated NK cells. Moreover, these cells showed a significant increase in degranulation capacity as well as cytokine secretion (IFN-Ƴ and TNF-α) throughout study period. In conclusion, we hypothesize that infusion of high-dose of ex-vivo activated NK cells could be associated with improvements of NK cell phenotype and function during immune reconstitution after allo-SCT.