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The study of genetic changes of children patients suffering from the acute lymphoblastic leukemia (ALL) using mFISH / mBAND and micro-arrays.
Bártů, Linda ; Zemanová, Zuzana (advisor) ; Březinová, Jana (referee)
Acute lymphoblastic leukemia is the most common malignancy in children. The most important examination at the time of diagnosis includes karyotype of leukemic cells which divides patients into prognostic groups according to cytogenetic finding. In up to 90 % of patients the chromosomal aberrations with well known clinical significance are designated. One of cytogenetic type is high hyperdiploid ALL (51-68 chromosomes) associated with favorable prognosis. Nevertheless, relapses of the disease occur even in these children. One possible reason why this happens could be an increased genomic instability of leukemic cells that causes cryptic structural rearrangements. In a retrospective study, we examined a total of 232 children with newly diagnosed B-ALL using conventional cytogenetic analyses and interphase fluorescence in situ hybridization (I-FISH) with a panel of DNA probes (Abbott Vysis) in order to detect heteroploid cells. In patients with suspect cryptic structural chromosome aberrations, we analyzed the karyotypes in detail by multicolor FISH and multicolor banding (mFISH/mBAND; MetaSystems). The extent of aberrations was determined by comparative genomic hybridization on BAC arrays (array CGH; BlueGnome). Cell clones with high hyperdiploid karyotype were detected in a total of 102 children (44 %). In...
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Unbalanced changes in cancer cells genome and its role in cancer pathogenesis
Lhotská, Halka ; Zemanová, Zuzana (advisor) ; Jarošová, Marie (referee) ; Kuglík, Petr (referee)
Malignant transformation of cell is characterized by genomic instability that involves unbalanced changes besides other things. We analyzed genomic aberrations, promoter methylation and mutations of several clinically relevant genes using I-FISH, mFISH, mBAND, CGH array, SNP array, MLPA, MS-MLPA and MS-PCR methods. We focused on two groups of patients well known for frequent appearance of unbalanced changes - patients with malignant brain tumors (gliomas) and patients with myelodyspastic syndromes (MDS). In patients with low grade glioma (WHO grade I - II), the codeletion of 1p/19q (82,6% oligodendrogliomas and oligoastrocytomas), mutation of IDH1/IDH2 genes (87% WHO grade I-II gliomas), copy neutral loss of heterozygozyty of 17p (72,2% astrocytomas) and higher presence of unbalanced aberration in astrocytomas belongs to the most frequent findings. We described yet unpublished methylation of MLH3 gene promoter in 60,9% oligodendrogliomas and in 27,3% astrocytomas. We also observed clonal evolution in patients with recurrent tumors. We studied secondary rearrangements of deleted chromosome 5 in patients with MDS and complex karyotype and we described its most recurrent translocation partners and breakpoints. We observed chromothripsis in 49% of these patients and it was frequently associated with...
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Aberrations of chromosome 5 in adult patients with myelodysplastic syndromes (MDS)
Šejgunovová, Nikola ; Zemanová, Zuzana (advisor) ; Urbánková, Helena (referee)
Myelodysplastic syndrome (MDS) is a clonal disease of hematopoesis resulting from damage to hematopoietic stem cells. The most common chromosomal aberration in patients with MDS is deletion of the long arms of chromosome 5, del(5q). The aim of this study is to analyse unbalanced aberrations of chromosome 5 in MDS patients, to compare the extent of 5q deletion in groups of patients with isolated del(5q) and with del(5q) in complex karyotypes, and to study the effect of the extent of del(5q) on overall survival and prognosis of the disease. We combined cytogenomic methods to examine 88 bone marrow samples from patients with MDS and del(5q) confirmed by conventional banding methods. Del(5q) was present in the karyotype as an isolated aberration in 31 patients (35,2 %), in combination with one other clonal aberration in 9 patients (10,2 %), and as part of complex karyotypes in 48 patients (54,6 %). Patients with complex karyotypes had a lower overall survival than patients with isolated del(5q). The occurrence of complex karyotypes was associated with a large extent of 5q deletion. When both the occurrence of complex karyotypes and the extent of 5q deletion were considered, only karyotype complexity had a significant effect on patients' overall survival. The extent of the deletion does not affect...
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Receptors involved in regulations of inflammatory reactions in renal diseases
Eis, Václav ; Mandys, Václav (advisor) ; Hes, Ondřej (referee) ; Zemanová, Zdeňka (referee)
Eis, V., Luckow, B., Vielhauer, V., Sireke, J., Linde, Y., Segerer, S., Perez de Lema, G., Cohen, C. D., Kretzler, M., Mack, M., Horuk, R., Murphy, P. M., Gao, J.-L., Hudkins, K. L., Alpers, C. E., Gröne, H.-J., Schlöndorff, D., Anders, H.-J.: Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction. J Am Soc Nephrol, 15, 2004; 2: 337-347. We examined the role of chemokine receptor CCR1 and CCR5 in renal inflammatory infiltrate and subsequent interstitial fibrosis. Unilateral ureteral obstruction model in mice deficient for CCR1 or CCR5 was used for experiments. Analysis of UUO kidneys from CCR1-deficient mice or BX471 treated mice revealed a reduction of interstitial macrophages and lymphocytes compared with wild type controls. In contrast, renal leukocytes and fibrosis were unaffected in CCR5-deficient mice with UUO. Interstitial fibroblasts, renal TGF-β1 mRNA expression, interstitial volume and collagen I depositions were significantly reduced in CCR1-deficient mice. Lack of CCR5 does not affect renal fibrosis after UUO. Thus, CCR1 but not CCR5 is required for leukocyte recruitment and fibrosis after UUO in mice. Anders, H.-J., Belemezova, E., Eis, V., Segerer, S., Vielhauer, V., Perez de Lema, G., Kretzler, M., Cohen, C. D.,...
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The role of molecular genetic and cytogenetic analyses in the diagnosis and prediction of treatment response in patients with non-Hodgkin lymphomas
Berková, Adéla ; Zemanová, Zuzana (advisor) ; Goetz, Petr (referee) ; Smolej, Lukáš (referee)
Malignant lymphoproliferative disorders include highly heterogeneous entities, i.e. lymphomas (Non-Hodgkin - NHL, as well Hodgkin's lymphoma), lymphoid leukemias, multiple myeloma and others. As currently many chromosomal aberrations with diagnostic and prognostic significance are known, molecular cytogenetic analyses of tumor cell genome has become a substantial examination also in lymphoproliferative disorders. This thesis focuses primarily on chronic lymphocytic leukemia (CLL), which is one of the mature B-cell neoplasms and represents the most common type of leukemia. We analyzed four most frequently found aberrations (13q14 deletion, ATM and TP53 gene deletion, and trisomy 12) by fluorescence in situ hybridization (FISH) and also IgH gene aberrations in some patients. We compared the findings with other factors and clinical characteristics. This work shows that the conventional G-banding is analysis relatively little relevant. FISH was more effective in detecting aberrations in CLL. Although none of the four aforementioned changes is specific to CLL, the prognostic impact is significant, particularly that of TP53 deletion. Next, detection of some IgH gene translocations is essential in differential diagnosis of CLL and other NHL (follicular, mantle cell, diffuse large B cell, Burkitt's...
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Clonal evolution of leukemic cells and its role in the progression of leukemia and preleukemia
Svobodová, Karla ; Zemanová, Zuzana (advisor) ; Urbánková, Helena (referee) ; Šubrt, Ivan (referee)
Clonal evolution is a multistep process characterized by progression of the disease, adverse prognosis and shortening of overall survival. The aim of the dissertation was a detailed characterization of identified changes in patients with myelodysplastic syndromes (MDS) and clonal evolution and evaluation of their prognostic impact. We performed detail cytogenomic analyses in 36/469 (8%) patients with confirmed linear clonal evolution. We described 57 primary abnormalities (32% MDS-specific) at the time of diagnosis, the most frequent was deletion of long arm of chromosome 5. We proved 156 secondary aberrations (21% MDS-specific) during the course of the clonal evolution, the most frequent were trisomies/tetrasomies of chromosome 8. We identified acquired uniparental disomies (aUPD) in 19% of patients. In MDS-specific aUPDs 4q, 11q and 17p, we proved homozygous mutations of TET2, c-CBL and TP53 genes. We found a statistically significant difference in overall survival between the groups of patients divided according to their diagnostic cytogenomic findings. In patients with clonal evolution before treatment 54% of aberrations were gains of whole chromosomes, by contrast 44% of abnormalities identified in patients with clonal evolution after treatment were monosomies or deletions. The study of clonal...
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Formation of complex chromosomal rearrangements in cancer cells and significance of these events
Rochlová, Kristina ; Zemanová, Zuzana (advisor) ; Rothová, Olga (referee)
Chromoanagenesis is a catch-all term of recently described catastrophic events that generate complex karyotypes. These events are divided according to the characteristic features and are termed chromothripsis, chromoplexis and chromoanasynthesis. Chromothripsis represents a disintegration of chromosomes or their parts into hundreds of small fragments. Those chromosome fragments are then incorrectly reassembled. Chromoplexis rearrangements are not very different from chromothripsis rearrangements. The main difference is a lower number of breakpoints and the distribution of aberrations in the whole genome. The erroneous replication processes occur during chromoanasynthesis. There are several mechanisms responsible for breakdowns of a DNA molecule. In the case of chromothripsis, micronucleus formation is probably the most important mechanism. During chromoplexis, transcriptional stress plays a major role. Replication stress is associated with chromoanasynthesis rearrangements. The result of all these processes are highly rearranged chromosomes with numerous losses or gains of genetic material. This work summarizes the current knowledge of the mechanisms that are mentioned above and the genesis of complex aberrations. At the same time, it represents the connection between complex karyotype and clonal...
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Frequency and significance of genetic changes in the genome of leukemia cells in children with T-ALL
Sládková, Lucie ; Zemanová, Zuzana (advisor) ; Březinová, Jana (referee)
T-ALL (T-cell acute lymphoblastic leukemia) is identified in 10-15 % cases of pediatric acute lymphoblastic leukemia and it is a clinically and genetically heterogeneous disease. About 50 % of patients have normal karyotype and although a number of cryptic recurrent chromosome aberrations have been reported their prognostic significance is not entirely clear. The aim of the study was to analyze bone marrow cells of children with T-ALL using cytogenomic methods to determine the frequency of cryptic aberrations and to assess their importance for disease prognosis. We examined diagnostic samples of 67 children with T-ALL (19 girls and 48 boys, median age 8 years). We analyzed the changes by G- banding, I-FISH (Dako, Abbott) and MLPA (MRC-Holland) methods. We detected cryptic aberrations in 60 children (91 %). The most frequent changes were deletions of the CDKN2A gene (48×) which were usually observed in combination with other changes and aberrations of loci for TCR genes (20×). TLX3 gene rearrangements were detected in 18 cases and were never associated with rearrangements of TCR loci. Complex karyotype was detected in 10 patients with recurrent breakpoints 5q35 and 10q24. 45 patients live in the first or second complete remission, relapse occurred in 14 children and 20 died. Statistical analysis of...
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MicroRNAs in AML pathogenesis
Koutová, Linda ; Korabečná, Marie (advisor) ; Zemanová, Zuzana (referee) ; Kotyza, Jaromír (referee)
Acute myeloid leukemia (AML) is a very heterogeneous disease associated with cytogenetic aberrations and genetic mutations. Many of these changes have been revealed and their detection became usual part of the diagnostic process today. However, changes of expression profiles of small, noncoding RNAs, so called microRNAs (miRNAs), are less known and not used for diagnostics yet. These RNAs, 19-24 nucleotides long, take part in the regulation of expression of different genes through complementary base pairing to the 3'non- translated region (3'UTR) of the target messenger RNA (mRNA). They can influence key processes of the cell, like differentiation, proliferation or apoptosis. The changes in expression of different miRNAs are known from different types of cancers. In solid tumors, they are usually detected from bioptic samples; but also plasma samples are now in the center of attention as so called liquid biopsies providing the information about molecular genetic events in the organism. Many studies have revealed deregulated miRNAs in the bone marrow, full blood or isolated progenitor cells (CD34+) of AML patients, only four of them have analyzed plasma samples. We focused on the plasma samples and we targeted on such miRNAs, which levels differ at AML diagnosis and after the chemotherapy. Out of...
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