Wilms Tumour (WT) is one of the commonest tumours in children. Hepatic Sinusoidal Obstruction Syndrome (HSOS) is a documented complication following treatment of WT. The role of malnutrition in the development of HSOS has not been studied. Malnutrition reduces tolerance to chemotherapy and shows increased risk for toxicity. Purpose of study To determine the prevalence of HSOS in children with WT, as well as its predisposing factors and outcomes. Method A descriptive retrospective analysis of medical records of children treated for WT, who developed HSOS, at the Paediatric Haematology/Oncology Unit, Chris Hani Baragwanath Hospital. Results 82 patients were evaluated. 19 (23%) showed features compatible with HSOS. Younger age, irradiation and a right-sided WT predicted the development of HSOS but were not statistically significant. Serum albumin levels were lower in the affected group (P = 0.02). Apart from 2 deaths, outcomes were good, with patients showing full resolution of symptoms. Conclusion A higher prevalence of HSOS was shown than previously reported. Low serum albumin levels points to the role of malnutrition. Effort needs to be put into the various methods of identifying malnutrition. Long term follow-up is needed.

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Das ADAMTS16-Gen kodiert für eine Metalloproteinase, deren Funktion und Regulation bislang nicht beschrieben sind. Die ADAMTSs werden von Zellen verschiedener Organsysteme sezerniert und sind für den Abbau extrazellulärer Matrixbestandteile und die Prozessierung von Oberflächenrezeptoren, Signalmolekülen oder Wachstumsfaktoren verantwortlich. In der vorliegenden Arbeit wurden die gewebespezifische Lokalisation von Adamts16 und die möglichen Funktionen der Metalloproteinase im Urogenitalsystem untersucht. Weiterhin konnte die Regulation der Adamts16-Expression durch das Wilms-Tumor Protein beschrieben werden. In verschiedenen Zelllinien des Urogenitalsystems konnte eine Wt1-abhängige Adamts16-Expression festgestellt werden. Zudem erfolgte im Urogenitalsystem eine Koexpression von Adamts16 und Wt1 in embryonalen und adulten Podozyten, somatische Zellen der XX-Gonadenanlage und Granulosazellen und Epithelzellen des adulten Ovars. Im Testis war Adamts16 ohne signifikante Wt1-Koexpression in Spermatozyten und elongierten Spermatiden lokalisiert. Außerhalb des Urogenitalsystems waren Adamts16 und Wt1 im Epikard koexprimiert. Ein Wt1-Knockdown in Epikardzellen und embryonalen Nieren zeigte jeweils einen Rückgang des Adamts16-Expressionsniveaus. Ein Adamts16-Knockdown in embryonalen Nieren resultierte in verminderten Ureterverästelungen, was eine funktionelle Rolle von Adamts16 in der murinen Nierenentwicklung ex vivo andeutet. Der Wt1-Knockdown in Gonadenkulturen zeigte, dass Wt1 die Adamts16-Expression in XY-Gonaden hemmt, in XX-Gonaden hingegen aktiviert. Innerhalb des Adamts16-Gens konnten drei Wt1-Konsensusmotive identifiziert werden. Mit Hilfe von EMSAs und ChIPs konnte die Bindung der Wt1(-KTS)-Isoform an diese Konsensusmotive belegt werden. Ein Reportergenassay zeigte die Aktivierung des Adamts16-Promotors durch Wt1(-KTS) in Granulosazellen, wobei eine Verkürzung der Adamts16-Promotorsequenz zu einer Reduktion der Promotoraktivität führte. / The Adamts16 gene encodes for a metalloproteinase, whose function and regulation is hardly explored. ADAMTSs are secreted by different cells of various organs and are responsible for breaking down extracellular matrix compounds and processing signaling molecules, growth factors and surface receptors. In this work the tissue specific localization of Adamts16 and its possible function and regulation within the genito-urinary system were analyzed. Furthermore the regulation of Adamts16 through the wilms tumor transcription factor Wt1 is described. Different cell lines derived from the genito-urinary system showed a Wt1-dependent mRNA expression of Adamts16. In addition both proteins were co-expressed in embryonic and adult podocytes, somatic cells of the embryonic XX-gonad and granulosa and epithelial cells of the adult ovary. The testes showed a Wt1-independent Adamts16 expression in spermatocytes and elongated spermatids. Outside the genito-urinary system Adamts16 and Wt1 were co-expressed in the epicardium. Knockdown of Wt1 in both epicardial cells and embryonic kidney explants showed a decrease in the Adamts16 mRNA expression level. In turn the Knockdown of Adamts16 led to an inhibited branching morphogenesis in embryonic kidney explants. This indicates a functional role of Adamts16 in the ex vivo kidney development. Knockdown of Wt1 in cultured embryonic gonads revealed that Wt1 inhibits the expression of Adamts16 in XY-gonads but activates it in XX-gonads. Three Wt1 consensus motives were identified within the Adamts16 gene. Using EMSA and ChIP the binding of the Wt1(-KTS)-isoform to all three consensus motives was verified. The ability of Wt1 to activate the Adamts16 promoter was confirmed through reporter gene assays in granulosa cells.

Les leucémies aiguës myéloïdes (LAM) sont des hémopathies malignes hétérogènes déclenchées, dans la plupart des cas, par des anomalies génétiques (mutations, translocations ou inversions). Elles se caractérisent par un blocage de la différenciation de certains progéniteurs ou précurseurs hématopoïétiques (blastes) et leur prolifération clonale incontrôlée provoquant leur accumulation dans la moelle osseuse. Le traitement actuel de ces patients repose essentiellement sur l’utilisation d’agents de chimiothérapie (cytarabine associée à une anthracycline) permettant d’éliminer les cellules leucémiques et d’obtenir une rémission complète (RC) (définie morphologiquement comme une moelle osseuse normale avec moins de 5% de blastes). Cette RC est obtenue chez une majorité des patients mais plus d’un patient sur deux va rechuter quelques mois après l’arrêt du traitement. Ces rechutes attestent de la persistance de cellules leucémiques résiduelles après le traitement, que l’on appelle maladie résiduelle mesurable (MRD). Celle-ci a été mise en évidence grâce au développement de technologies performantes et sensibles tels que la cytométrie en flux multi-paramétrique et la PCR en temps réel (qPCR) permettant ainsi la détection de profils d’expression ou d’anomalies génétiques associés aux LAM. A ce jour, plusieurs mécanismes ont été décrits pour expliquer la présence de cette MRD. Celle-ci peut être causée par une résistance au traitement de certains sous-clones leucémiques (anomalies génétiques intrinsèques leur conférant une résistance ou un phénotype quiescent) ou par la présence de cellules souches leucémiques (naturellement quiescentes). Le système immunitaire pourrait également jouer un rôle en induisant la quiescence de certaines cellules les rendant résistantes aux chimiothérapies conventionnelles, ou en contrôlant leur croissance tumorale par l’établissement d’un état d’équilibre entre leur prolifération et leur lyse. Les modèles murins de LAM actuellement utilisés permettent d’étudier la leucémogenèse et l’efficacité thérapeutique de certains composés mais font abstraction du rôle de la réponse immunitaire dans ces processus du fait de leur immunodéficience. De plus, aucun modèle murin de MRD leucémique n’existe pour étudier les causes de la persistance cancéreuse après traitement par chimiothérapie. Ainsi, le but de cette thèse a été de développer un modèle murin syngénique et immunocompétent de MRD leucémique sur-exprimant ou non le gène Wilms’ Tumor 1 (WT1). WT1 est un des rares antigènes décrits dans les LAM et une réponse lymphocytaire cellulaire et humorale dirigée contre cette protéine a été décrite chez ces patients. La création de ce modèle sur-exprimant ou non WT1 permettra ainsi d’étudier le rôle de la réponse immunitaire spécifique de celui-ci dans la persistance leucémique. Pour développer ce modèle nous avons, dans un premier temps, caractérisé phénotypiquement et génotypiquement des sous-clones isolés de la lignée leucémique C1498 capable d’induire une LAM de type myélo-monocytaire chez des souris immunocompétentes C57BL/6J. Dans un deuxième temps, certains sous-clones ont été sélectionnés pour leur sensibilité à la cytarabine et transfectés de manière à exprimer stablement une protéine fluorescente (ZsGreen) en association ou non avec la protéine WT1. Enfin, ce modèle de MRD leucémique a été obtenu en modulant la quantité de cellules leucémiques injectée ainsi que la cinétique et la dose d’injection de la cytarabine. La MRD a été suivie par cytométrie en flux (expression ZsGreen) et par qPCR (expression ZsGreen et/ou de Wt1) dans le sang et la moelle osseuse des souris survivantes grâce au traitement [...]. / Acute myeloid leukemia (AML) is a genetic disorder leading to a blockade of differentiation and a clonal expansion of hematopoietic progenitors or precursors (called blasts) which accumulate in the bone marrow and then invade the blood stream. Conventional treatment relies on the use of chemotherapy agents (cytarabine in combination with an anthracycline) to eliminate leukemia cells and achieve complete remission (defined as normal bone marrow morphology with less than 5% blasts). This complete remission is achieved in a majority of patients but more than 50% of them will relapse several months after the treatment. These relapses indicate the presence of residual leukemic cells after treatment, known as measurable residual disease (MRD). It has been highlighted by the development of efficient and sensitive molecular biology technologies such as multi-parameter flow cytometry and real-time PCR allowing the detection of AML-associated expression patterns and genetic abnormalities. Several mechanisms have been described that can explain the presence of this MRD. It may be caused by the resistance to treatment of certain leukemic sub-clones (resistance-conferring mutations or quiescent phenotype) or the presence of leukemic stem cells. Finally, the immune system could also induce the quiescence of certain leukemic cells rendering them resistant to conventional chemotherapies, or control their growth leading to a state of equilibrium between their proliferation and lysis. Several AML mouse models allow the study of leukemogenesis and the testing of new therapeutic agents for leukemic cells eradication. However, they are mostly based on the transfer of human leukemic cells in immune-deficient mice and do not provide information about the role of the immune system in the leukemic cell survival, sub-clonal expansion or persistence. Moreover, there is still no available leukemia MRD mouse model allowing the study of leukemic cell persistence after chemotherapy treatment. According to these findings, the aim of this thesis was to develop a syngeneic and immune-competent mouse model of leukemia MRD overexpressing or not the Wilms' Tumor 1 (WT1) gene. The WT1 protein is described as an antigen associated with AML and is targeted by specific lymphocyte cellular and humoral responses in AML-affected patients. Creating a syngeneic and immune-competent leukemia MRD mouse model overexpressing or not this antigen will allow determining the role of this specific immune response in the cancer cell persistence. To set up this model, we first phenotyped and genotyped sub-clones isolated from the murine C1498 leukemic cell line able to induce a myelo-monocytic AML in immune-competent C57BL/6J mice. In a second step, certain sub-clones were selected for their sensitivity to cytarabine treatment and transfected to stably express the fluorescent ZsGreen protein with or without the WT1 antigen. Lastly, the MRD mouse model was obtained after modulation of various parameters such as the amount of leukemic cells administered, the kinetics and injection doses of chemotherapy. The leukemia MRD was monitored by flow cytometry (expression of the ZsGreen protein) and by real-time PCR (expression of the ZsGreen and/or Wt1 genes) in the peripheral blood and the bone marrow of treated and surviving mice. Thus, we generated a syngeneic and immune-competent leukemia MRD mouse model useful to study the immune mechanisms involved in the persistence of leukemic cell after treatment and to test new (immune)-therapeutic strategies targeting these residual cells.

Wilms’ tumor gene 1 (WT1) is a zinc finger transcriptional regulator with crucial functions in embryonic development. Originally WT1 was described as a tumor suppressor gene, but later studies have shown oncogenic properties of WT1 in a variety of tumors. Because of its dual functions in tumorigenesis, WT1 has been described as a chameleon gene. In this thesis, the significance of WT1 as a biomarker was investigated in acute myeloid leukemia (AML), clear cell renal cell carcinoma (ccRCC), ovarian carcinoma (OC) and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Previous studies have suggested that expression of WT1 is a potential marker for detection of minimal residual disease (MRD) in AML. We aimed to define expression of WT1 as an MRD marker in AML. In adult AML patients, we found that a reduction of WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis was associated with an improved overall survival (OS) and freedom from relapse (FFR). In peripheral blood, a reduction of WT1 expression (≥ 2-log) detected between 1 and 6 months after treatment initiation was associated with an improved OS and FFR. WT1 harbor pathogenic genetic variants in a considerable proportion of AML and T-lymphoblastic leukemia (T-ALL), but mutations have not been reported in BCP-ALL. We aimed to evaluate the clinical impact of WT1 mutations and single nucleotide polymorphisms (SNPs) in BCP-ALL. Pathogenic mutations in the WT1 gene were rarely seen in childhood BCP-ALL. However, five WT1 SNPs were identified. In survival analyses, WT1 SNP rs1799925 was found to be associated with worse OS, indicating that WT1 SNP rs1799925 may be a useful marker for clinical outcome in childhood BCP-ALL. We also explored whether WT1 mutations and SNPs in ccRCC could be used as biomarkers for risk and treatment stratification. We therefore examined whether SNPs or mutations in WT1 were associated with WT1 expression and clinical outcome. Sequencing analysis revealed that none of the previously reported WT1 mutations were found in ccRCC; however, we identified six different WT1 SNPs. Our data suggest that pathogenic WT1 mutations are not involved in ccRCC, and the prognostic significance of WT1 SNPs in ccRCC is considerably weak. However, a favorable OS and disease-specific survival were found in the few cases harboring the homozygous minor allele. OC has a poor prognosis, and early effective screening markers are lacking. Serous OCs are known to express the WT1 protein. Overexpressed oncogenic proteins can be considered potential candidate antigens for cancer vaccines and T-cell therapy. It was therefore of great interest to investigate whether anti-WT1 IgG antibody (Ab) measurements in plasma could serve as biomarkers of anti-OC response. We found limited prognostic impact, but the results indicated that anti-WT1 IgG Ab measurements in plasma and WT1 staining in tissue specimens could be potential biomarkers for patient outcome in the high-risk subtypes of OCs. In conclusion, the results of this thesis indicate that WT1 gene expression can provide information about MRD of patients with AML, and WT1 SNP rs1799925 may be used as a biomarker for predicting clinical outcome in childhood BCP-ALL. In ccRCC, the prognostic significance of WT1 SNPs is weak and limited to the subgroup of patients that are homozygous for the minor allele. In OCs anti-WT1 IgG Ab measurement in plasma and WT1 staining in tissue specimens could possibly be used as biomarkers for predicting patient outcome in the high-risk subtypes of OCs.

Myeloid leukemias include malignant diseases characterized by clonal expansion of the myeloid cell lineage. While in case of chronic myeloid leukemia (CML), the main cause of the disease has already been identified - t(9;22) and the aktivity of the fusion product of the translocation BCR-ABL, acute myeloid leukemia (AML) has been associated with plenty of different translocations and mutations. The aim of this work was to contribute to the improvement of monitoring of patients with myeloid leukemias via detailed study of the panleukemic marker Wilms tumor gene 1 (wt1) expression. Prognostic value of wt1 expression has been proved for AML patients, however, it has not yet been confirmed for CML patients. Expression of different wt1 variants (more then 36 protein products) is known very poorly in both, AML and CML as well as in normal hematopoiesis. Most of the study is focused on CML, only limited parts are dedicated to AML. In the first part of the work, we clearly proved prognostic value of total wt1 mRNA expression for CML patients. Statistical evaluations revealed critical wt1 values which enable to specify prognosis of patients responding non-optimally to imatinib. Bcr-abl looses much of its prognostic value in these patients. Further, we have designed and optimized PCRs for selected wt1...

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Nephroblastoma is the most prevalent pediatric kidney tumor, which occurs primarily in younger children with the average age at diagnosis of 42,5 months for girls and 36,5 months for boys. Even though its treatment is currently very succesful and the overall survival rate reaches over 90 %, there are still more things to be discovered and improved. An important role for the right choice of treatment plays not only the histology of tumor, but also the chromosomal changes present at tumor. Some of them (for example 1q gain, simultaneous deletion of 1p and 16q, TP53 deletion) were confirmed as negative prognostic markers because they are associated with an increased risk of relapse or with anaplastic type of nephroblastoma that is included in a high risk group. These changes are therefore used together with the tumor histology for stratification of nephroblastomas. Some of these changes were found in a heterogeneous state (only in a part of the cells) in nephroblastoma, which also complicates the treatment of the patient and which cannot be solved when only one sample is taken from the tumor. In this work we concentrated on the detection of chromosomal changes present in nephroblastomas of 44 patients and their associations with clinical data. We have proved some of the known associations (22q...

Orientadores: Laurecir Gomes, Maria Tereza Cartaxo Muniz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T03:40:26Z (GMT). No. of bitstreams: 1 Arruda_IzabelBarrosde_D.pdf: 1365851 bytes, checksum: 2a1a28a0a33d73a75e48051e8b746627 (MD5) Previous issue date: 2014 / Resumo: O tumor de Wilms é uma neoplasia embrionária, originada de células do blastema metanéfrico, sendo histologicamente caracterizado como tumor trifásico, pois as células blastematosas, estromais e epiteliais estão presentes em proporções variáveis, com grande diversidade de arranjo celular e graus de diferenciação com ou sem anaplasia. Este tumor está associado a síndromes genéticas e a malformações do trato geniturinário. O objetivo desta tese foi avaliar as alterações nos genes WT1 e WT2 e a relação do índice apoptótico com o estadiamento tumoral. Para investigação das alterações nos genes WT1 e WT2 foi utilizada o método FISH, a imunorreatividade das proteínas WT1, p53 e MMP-2 foi empregada imunoistoquímica e para detectar células em apoptose, utilizou-se o método TUNEL. Os resultados evidenciaram cópias extras dos genes WT1 e WT2, com citogenética molecular nuc ish (WT1x3),(WT2x3) [100%], sugerindo que a evidencia da cópia-extra do gene WT2 pode ser explicada por trissomia ou por isodissomia uniparental, porém a presença da cópia-extra do WT1, até o momento, não foi relatada em qualquer caso clínico na literatura, sendo necessários mais estudos que a justifiquem. Por imunohistoquímica, constatou-se intensa imunomarcação no tumor para WT1 (80%), p53 (96,4%), assim como no estroma e ao redor do tumor, para MMP-2 (97%). Possivelmente essa alta expressão de WT1 pode estar refletindo a perda da regulação desse gene no desenvolvimento normal celular. A detecção da p53 neste estudo pode indicar a mutação do gene TP53, sugerindo que os tumores de Wilms poderiam progredir para anaplasia, após adquirem mutação do TP53, porém não como um evento isolado. Outros eventos seriam necessários para esta progressão, como a perda do alelo normal do TP53. A alta expressão da MMP-2 no estroma e ao redor do tumor não implica em sua atividade proteolítica, já que essa proteína pode estar em sua forma latente como também ativa e a técnica imunohistoquímica utilizada neste trabalho não diferencia essas formas. Não se identificou diferença significativa do índice apoptótico segundo estadio tumoral (p=0,937), mas os tumores em estadio III apresentaram índice seis vezes menor o daqueles em estadio II, em ausência de neoadjuvância, proporção que aumentou para 29 vezes, na presença de quimioterapia neoadjuvante, parecendo indicar que a neoadjuvância pode interferir sobre os mecanismos envolvidos na apoptose / Abstract: Wilms' tumor is an embryonic neoplasm arising from cells of the metanephric blastema, being histologically characterized as three-phase tumor, because blastematosas, stromal and epithelial cells are present in varying proportions, with great diversity of cell arrangement and degree of differentiation with or without anaplasia. This tumor is associated with genetic syndromes and malformations of the genitourinary tract. The aim of this thesis was to evaluate changes in WT1 and WT2 genes and the ratio of apoptotic cells to tumor staging. For investigation of changes in both WT1 and WT2 genes, the FISH method was used for immunoreactivity of the WT1 p53 and MMP-2 protein was employed and immunohistochemistry to detect cells undergoing apoptosis, we used the TUNEL. The results showed extra copies of WT1 and WT2 genes, with molecular cytogenetic nuc ish (WT1x3), (WT2x3) [100%], suggesting that the evidence of extra copy of WT2 gene can be explained by trisomy or uniparental isodisomy, but the presence of extra copy of WT1, so far, has not been reported in any clinical case in the literature, being necessary more studies to justify it. By immunohistochemistry, intense immunostaining was found in the tumor WT1 (80%), p53 (96.4%) as well as in the stroma surrounding the tumor, MMP-2 (97%). Possibly this high expression of WT1 may reflect the loss of regulation of this gene in the normal cell development. The detection of p53 in this study may indicate the mutation of the TP53 gene, suggesting that the Wilms' tumors may progress to anaplasia, after acquiring mutation of TP53, but not as an isolated event. Other events are needed for this progression, as loss of the normal allele of TP53. The high expression of MMP-2 in the stroma and around the tumor does not imply its proteolytic activity, since this protein may be at its latent form as well as active and the immunohistochemical technique used in this study does not differentiate these forms. There was no significant difference in the apoptotic index according to the tumor stage (p = 0.937), but in stage III tumors showed rate six times smaller than those in the stage II, in the absence of neoadjuvant therapy, rising to 29 times in the presence of chemotherapy neoadjuvant, seeming to indicate that neoadjuvant therapy may interfere with the mechanisms involved in apoptosis / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

Wilms tumör (WT) är en malign snabbväxande tumör och den vanligaste solida buktumören hos barn under sex år. En kombination av operation, cytostatika och strålbehandling har lett till att 90 % av barnen idag blir helt botade. Enligt ett enhetligt europeiskt morfologiskt klassifikationssystem och behandlingsprotokoll behandlas alla barnpatienter med WT med cytostatika pre-operativt innan nefrektomi. Den histologiska tumörtypen, klassad efter operation, är helt avgörande för vidarebehandling av WT. Tumören består av tre celltyper, stromala epiteliala och blastemala, där de blastemala har en stor likhet med mesenkymala celler i njuren hos embryot. Tumörer med mer än 2/3 blastem klassificeras som högrisktumörer som följs upp med ytterligare, extra intensiv kemoterapi. I dagsläget görs en histologisk bedömning av tumörens riskgrupp endast på hematoxylin-eosin färgning. Proteinet SIX1 har nyligen rapporterats fungera som lämplig biomarkör för blastem. Med hjälp av immunohistokemi undersöktes i detta arbete om SIX1-färgning på blastem kan tillämpas kliniskt för säkrare riskbedömning av WT. Efter omfattande utvecklingsarbete med testning av olika varianter av förbehandling och detektionsmetod kunde ett fullgott protokoll för kliniskt bruk utformas. Detta kommer nu att implementeras inom klinisk patologi i Skåne. / Wilms tumor (WT) is a malign fast growing tumor and the most common solid abdominal tumor amongst children under the age of 6. A combination of surgery, chemotherapy and radiotherapy has led to a 90 % cured rate among children affected by WT. According to a uniform European morphological classification system and treatment protocol, all WTs receive pre-operative chemotherapy prior to nephrectomy. Histological tumor type, determined after surgery, is crucial for further treatment of WT. The tumor consists of three cell types: stromal elements, epithelial elements and blastema, of which the blastema has a great similarity to embryonic mesenchymal cells. Tumors that consist of more than 2/3 blastema are classified as high risk tumors, recieving additional intensive chemotherapy. Today, an assesment of WT histology is performed on hematoxylin-eosin stained specimens only. Recently, the SIX1 protein has been reported to function as a suitable biomarker for blastema. Using immunohistochemistry, it was investigated if SIX1 detection could be applied clinically for classification of WT. After several modifications of pre-treatment and primary antibody detection methodology, a clinically robust immunohistochemical staining protocol was finally developed. This will now be implemented in the clinic.