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Early Detection of Disease Progression in Patients with Myelodysplastic Syndromes.
Kaisrlíková, Monika ; Beličková, Monika (advisor) ; Kalinová, Markéta (referee) ; Zemanová, Zuzana (referee)
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders with a risk of transformation into acute myeloid leukemia (AML). The International Prognostic Scoring Systems integrate clinical data and cytogenetics to determine the risk of AML transformation for individual patients. Precise risk assessment is crucial for treatment decision- making. The aim of this thesis was to identify molecular markers for the early detection of disease progression in MDS patients. Using cDNA microarrays and next-generation sequencing, we targeted long noncoding RNAs (lncRNAs) and recurrently mutated genes in bone marrow cells. In addition, we focused on the identification of pathways related to the progression of MDS and understanding how the identified biomarkers participate. In the transcriptome study, we identify 4 candidate lncRNAs that may serve as prognostic biomarkers of the adverse course of MDS: H19, WT1-AS, TCL6, and LEF1-AS. Using various statistical approaches, we determined the level of H19 to be a strong independent prognostic marker. Furthermore, our data showed that disruption of transcriptional coregulation of the imprinting locus H19/IGF2 and miR-675, which directly regulates H19 and plays a role in tumorigenesis, accompanies disease progression. In the genomic study...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Differentiation plasticity of hematopoietic cells
Polgárová, Kamila ; Stopka, Tomáš (advisor) ; Otáhal, Pavel (referee) ; Šálek, Cyril (referee)
Hematopoiesis has been for many years seen as a straightforward process based on sequential restriction of cell fate potential leading to production of mature blood cells. In the last decade, however, several works documented an unexpected plasticity of hematopoietic cells with expanded potential of myeloid development from lymphoid progenitors and vice versa. Under physiologic conditions hematopoiesis is tightly controlled and the definite cell fate is denominated by multiple factors that all lead to changes in regulatory networks that include transcription factors, epigenetic changes and post-transcriptional modulations. Any disruption of this strict regulation, caused by mutations or other events, affects the proliferation and lineage fidelity of hematopoietic precursors. This may lead to clonal growth of variable significance or leukemogenesis and may possibly affect the treatment sensitivity of the hematological malignancies. For better understanding of hematopoietic regulation we described gene expression changes during physiological development of lymphoid and myeloid lineages and in leukemic specimens using our own simplified real-time PCR based platform. We investigated expression of 95 genes connected with lymphoid and myeloid differentiation or with leukemogenesis in sorted hematopoietic...
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The role of DNA repair mechanisms in the pathogenesis of myelodysplastic syndrome.
Válka, Jan ; Čermák, Jaroslav (advisor) ; Pospíšilová, Dagmar (referee) ; Penka, Miroslav (referee)
Background: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests the involvement of DNA repair mechanism defects in the pathogenesis of this disorder. The first part of this work was focused on monitoring of gene expression of DNA repair genes in MDS patients and on their alterations during disease progression. In the second part, next generation sequencing was used to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with MDS development was evaluated. Methods: Expression profiling of 84 DNA repair genes was performed on bone marrow CD34+ cells of patients with MDS. Screening cohort consisted of 28 patients and expression of selected genes was further validated on larger cohort of 122 patients with all subtypes of MDS. Paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 recombinase protein was done on samples acquired by trephine-biopsy. Targeted enrichment resequencing of exonic parts of 84 DNA repair genes was performed on the screening cohort of MDS patients. Real-time PCR was used for genotyping of selected SNPs in the population study. Results: RAD51 and XRCC2 genes showed...
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The role of cereblon in lenalidomide therapy of del(5q) myelodysplastic syndrome
Bokorová, Radka ; Fuchs, Ota (advisor) ; Lukačková, Renata (referee) ; Krijt, Jan (referee)
Myelodysplastic syndrome (MDS) with deletion of the long arm of the chromosome 5 (5q - syndrome, del( 5q)) can be characterized by anemia, macrocytosis, a normal or high platelet count, and hypolobulated megakaryocytes in the bone marrow. 5q - syndrome belongs to low - risk MDS, which means low risk to transform to acute myeloid leukemia. 5q - syndrome is ass ociated with female predominance and older age. Another sign is transfusion burden that is treated by erythropoiesis - stimulating agents (ESA) as erythropoietin (EPO). Moreover, the response of MDS patients is around 30 - 60% with the median of the response b eing ~ 24 months. The second line of treatment is lenalidomide (LEN) which is a derivate of teratogenic analog thalidomide. LEN increases erythropoiesis and inhibits the growth of del(5q) erythroid progenitors in vivo and it does not have a significant effe ct on the growth of normal CD34+ progenitors or cytogenetically normal progenitors in MDS with del(5q) clones. LEN is used as therapy in multiple myeloma, myelodysplastic syndrome, and lymphoma. LEN is an expensive agent and not every MDS patient re sponds to this therapy. This is a reason why is a need to find a biomarker for the determination of successful treatment. Some multiple myeloma studies showed that cereblon can be the biomarker...
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Myelodysplastic syndromes - search for the molecular basis]
Beličková, Monika
Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem cell disorders with ineffective hematopoiesis. It is characterized by morphological dysplasia, peripheral cytopenias affecting one or more cell lineages and an increased risk of transformation into acute myeloid leukemia (AML). The early stages of MDS can be considered a premalignant disease. The pathogenesis of MDS has not been fully explained yet, but due to the development of molecular genetic and cytogenetic methods, the origin and development of the disease is gradually being elucidated. In addition to the cytogenetic changes that are part of the prognostic system (IPSS-R), the somatic mutations found in different genes come to the forefront of interest. However, they are not routinely used in clinical practice. One of the objectives of this study was monitoring of mutations in TP53 gene in lower-risk MDS patients who generally have a good prognosis and for whom these findings have a particularly relevant prognostic significance. We investigated a total of 154 patients with lower-risk MDS, and 13% of them had a mutation. After dividing patients according to the presence of del(5q), we observed significant differences in the incidence of the mutations. The mutations were detected in 23.6% of patients with...
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Pathophysiologic aspects of myelodysplastics syndromes in relation to the effect of targeted imunomodulation and demetylation therapy
Jonášová, Anna ; Stopka, Tomáš (advisor) ; Maisnar, Vladimír (referee) ; Faber, Edgar (referee)
Myelodysplastic syndromes (MDS) represent a group of clonal stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenia, morphological dysplasia and the risk of transformation to acute myeloid leukemia (AML). MDS belongs to one of the most common hematological diseases in patients over 60 years old. MDS incidence is still increasing. Appropriate therapy of MDS remains challenging. There is no curative approach besides peripheral stem cells transplantation, which is regretfully appropriate only for a small group of patients due to a higher median age of the MDS population. This is why the search for therapeutic alternatives remains paramount. MDS treatment was rather frustrating until the recent introduction of two new therapeutic approaches: immunomodulation therapy with lenalidomide and epigenetic or demethylating therapy with 5-azacytidine. Both new drugs have significantly higher effect than standard therapy. However, the precise mechanism of this effect remains unknown. As a result, we decided to initiate several research projects while introducing this promising treatment to our patients. Our aim is to investigate the mechanism of both agents in relation to disease pathogenesis by examining changes of certain occurrences and factors prior to and during the course of...
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Differentiation plasticity of hematopoietic cells
Polgárová, Kamila ; Stopka, Tomáš (advisor) ; Otáhal, Pavel (referee) ; Šálek, Cyril (referee)
Hematopoiesis has been for many years seen as a straightforward process based on sequential restriction of cell fate potential leading to production of mature blood cells. In the last decade, however, several works documented an unexpected plasticity of hematopoietic cells with expanded potential of myeloid development from lymphoid progenitors and vice versa. Under physiologic conditions hematopoiesis is tightly controlled and the definite cell fate is denominated by multiple factors that all lead to changes in regulatory networks that include transcription factors, epigenetic changes and post-transcriptional modulations. Any disruption of this strict regulation, caused by mutations or other events, affects the proliferation and lineage fidelity of hematopoietic precursors. This may lead to clonal growth of variable significance or leukemogenesis and may possibly affect the treatment sensitivity of the hematological malignancies. For better understanding of hematopoietic regulation we described gene expression changes during physiological development of lymphoid and myeloid lineages and in leukemic specimens using our own simplified real-time PCR based platform. We investigated expression of 95 genes connected with lymphoid and myeloid differentiation or with leukemogenesis in sorted hematopoietic...
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