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Acute lymphoblastic leukaemia (ALL) is the most frequent childhood malignancy. One of the recent improvements in ALL treatment was the introduction of minimal residual disease (MRD) monitoring that enables risk stratification based treatment adaptation. The same MRD monitoring helps to choose relapse treatment, to guide indication for stem cell transplantation (SCT) and allows for a more personalized management of patients undergoing SCT. One of the main routes of MRD levels detection is characterisation of leukemic blasts using flow cytometry. However, flow cytometry is limited by its mainly manual expertise-based analysis. Such analysis is subjective and clearly insufficient for current complex data. While new computational tools are available for multidimensional flow cytometry data, there is an urgent need to test and adapt them for the use in clinical environment. The goal of this thesis is to detect immunophenotypes associated with leukaemia and their development by leveraging machine-assisted analysis of a set of diagnostic files selected based on information about more than three hundred thousand of multiparameter flow cytometry datasets. Advanced bioinformatic tools will help to detect blast and healthy haematopoietic populations, to derive their immunophenotypes and to identify individual...

Acute lymphoblastic leukemia is the most common type of cancer in children. It is a very heterogenous disease in which many recurrent chromosomal abnormalities have been described. The most important chromosomal abnormalities associated with a good prognosis are t(12;21)(p13;q22) which result in ETV6/RUNX1 fusion and hyperdiploidy. On the contrary findings suggesting a poor prognosis are t(9;22)(q34;q11) leading to fusion gene BCR/ABL1, MLL rearrangements or hypodiploidy. Heteroploidy is one of the most frequent findings in childhood ALL. It is characterised by nonrandom gain or loss of chromosomes from diploid cells. One of the most important findings in childhood ALL is hyperdiploidy where a non-random gain of chromosomes is present. Hyperdiploidy has a favorable prognosis and the impact of additional structural aberations requires further research. Another prognostically important group of heteroploidy is hypodiploidy. It is a quite rare finding and has a very poor outcome. There are non-random acquired chromosome losses observed in hypodiploid cells. Hypodiploid cell line may be masked with a doubled hyperdiploid clone which makes it difficult to identify. Proper and early cytogenetical analysis of heteroploid cells is very important as it contributes assigning correct diagnosis and risk stratification,...

The classical scheme of hematopoiesis presumes early separation of lymphoid and myeloid precursors. Recently, more complex models are put forward, suggesting greater flexibility of hematopoiesis with progenitors sharing lymphoid and myeloid potential. Acute hybrid leukemia is a malignancy, in which it is not possible to assess unambiguously myeloid or lymphoid lineage of origin. The behaviour of those malignancies favors new models of hematopoiesis. Our work concentrated mainly on the research of childhood leukemias with lineage switch from lymphoid to myeloid lineage during induction treatment. Our task within this extensive project was to determine lineage assignment of leukemic blasts using detection of immunoglobulin and T-cell receptor gene rearrangements. We confirmed that myeloid cells derived during the treatment in all patients descend from the original lymphoid clone. We also investigated the expression of selected genes in those cases compared to common leukemia types. Lastly, we explored prognostic impact of TCR rearrangements (and thus lymphoid lineage commitment) in T-lineage leukemia.

Philadelphia (Ph) chromosome, as a result of reciprocal translocation, is in majority of cases connected to two types of leukaemia - chronic myelogenous (CML) and acute lymphoblastic (ALL). The translocation occurs within large intronic sequences of BCR and ABL genes. The breakpoints are specific for individual patient and may be used as a target for monitoring of leukemic burden (MRD, minimal residual disease) during the treatment. In general, MRD is an important prognostic factor, which influences the treatment intensity. Two standardized methods are currently used for its monitoring. The first one is based on the detection of clonal specific Immunoglobulin and/or T-cell receptor genes rearrangements (and thus cannot be used for CML cases) at the DNA level, the second one utilizes detection of the BCR/ABL fusion gene at the mRNA level. Our aim was to optimize and standardize the process to find individual patient breakpoints on Ph chromosome and to use it for MRD quantification. We found the breakpoint in 80 % cases. The MRD data from 15 patients obtained by our method were compared to the levels obtained by standard methods (Ig/TCR and BCR/ABL transcript quantification). In all but 1 patient we found significant discrepancies, raising the questions about leukemic origin and the most accurate method for...

The Philadelphia chromosome has been discovered in 1960. This chromosomal aberration was mistakenly associated only with chronic myeloid leukaemia (CML) for decade. However, this type of translocation including chromosomes 9 and 22 was found in patients with different type of neoplasia - acute lymphoblastic leukaemia (ALL). Different lineage involvement has been found in these two types of leukaemia. Whereas in Ph-positive ALL, the Philadelphia chromosome is restricted to the lymphoid lineage, in CML patients mostly myeloid cells are those being Ph-positive. Hence it seems quite trivial to distinguish between ALL and CML. But there is a phase of CML called lymphoid blast crisis which is indistinguishable from ALL. The possibility of distinguishing between CML in lymphoid blast crisis and ALL would inhere in determining myeloid lineage involvement. Actually it had been shown that some patients with Ph+ ALL have involved also a myeloid lineage. Different types of treating protocols are used in CML and ALL. In addition, prognoses for both types of leukaemia are different. Thus it is crucial to distinguish between this two disorders and revealing of any difference can impact the treatment outcome of above mentioned malignancies. Detection of minimal residual disease according to involvement of myeloid or CD34+...