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In this thesis, we aimed to define the processes initiating pediatric leukemia by studying mutation accumulation during embryonic and fetal development in non-cancerous stem cells. Moreover, we investigated the molecular consequences of chemotherapy of normal, non-cancerous cells. Characterizing somatic mutations in non-cancerous stem cells is challenging due to polyclonal composition of normal tissues and the high error rate of single cell sequencing techniques. Therefore, we applied throughout this thesis a clonal expansion culture approach together with whole-genome sequencing to identify the somatic mutations in single fetal stem cells. In the general introduction (chapter 1) we discussed that many pediatric leukemias have a prenatal origin. In chapter 2, we characterized somatic mutation accumulation in human fetal hematopoietic stem and progenitor cells (HSPCs) and intestinal stem cells (ISCs) to investigate the mutation rate and the active mutational processes that cause mutation accumulation in these cells during prenatal development. We performed mutational signature analyses to obtain insights in these processes because such signatures reflect mutational processes that have been active in the assessed cell. Moreover, we characterized mutation accumulation in HSPCs and ISCs derived from Down syndrome (DS) fetuses to obtain unique insights into the mechanisms that contribute to pediatric leukemia in individuals at risk. This chapter is followed by a review (chapter 3), in which we described possible mechanisms that underlie the increased leukemia risk in DS children from a somatic evolutionary view. According to this model, cancer is the consequence of inheritable phenotypic variation within cell populations, which are subjected to selective forces present within the different tissue contexts. This model suggests that cancer only arises when the oncogenic clones are present in the correct tissue context. In chapter 4 we generated phylogenetic lineage trees of fetal stem and progenitor cells of different tissues to characterize somatic mutation accumulation very early during embryonic development, before gastrulation. Moreover, we compared these early embryonic mutations with the culture-associated mutation burden of iPSCs generated of cells of the same fetuses. In chapter 5 we exposed the ISCs of the assessed fetuses to various chemotherapeutic drugs to examine the molecular consequences of normal, non-cancerous cells. DS-ALL patients experience increased treatment related side effects of chemotherapy treatment compared to non-DS ALL patients, suggesting that their normal cells show increase sensitivity to chemotherapeutic drugs. Therefore, we studied differences in sensitivity for several chemotherapies, used for the treatment of pediatric B-ALL, on normal ISCs derived from karyotypically normal and DS fetuses. In addition, we examined the mutational and transcriptional consequences of hydroxyurea and cytarabine on these cells. Finally, we placed our findings in broader perspective in the general discussion of this thesis (chapter 6). |
