O interesse científico em rearranjos gênicos relacionados com a etiogênese e progressão do câncer relaciona-se, principalmente, à descoberta da fusão BCR/ABL na Leucemia Mieloide Crônica, sendo que desde então, houve uma evolução no manejo dessa doença, instigando uma série de estudos correlatos em outras neoplasias. Essas pesquisas culminaram no encontro do primeiro rearranjo gênico em tumores sólidos, o gene de fusão TMPRSS2/ERG, envolvendo a região promotora do gene da serina protease, o TMPRSS2, e o gene da família de fatores de transcrição ETS, o ERG. Ele é específico de adenocarcinoma da próstata, o que o torna forte candidato a biomarcador e já demonstra exercer papel de destaque no manejo clínico do câncer de próstata (CaP), tal qual o exercido pela fusão BCR/ABL. Sua frequência têm se mostrado associada a diversos fatores, sobretudo à etnia de origem. Indivíduos portadores de CaP oriundos de diversos países já foram estudados quanto à frequência dessa fusão e o resultado é bastante diversificado. No Brasil, entretanto, ainda não há dados a respeito desse rearranjo, e este trabalho visa contribuir para a identificação da frequência da mesma e sua contribuição para o diagnóstico e o tratamento do CaP no país. Para tal, utilizamos mRNA de 20 indivíduos com CaP provenientes do serviço de atendimento do HCFMRP/USP, e por meio da técnica de RT-PCR, obtivemos o cDNA dos mesmos que foram investigados quanto à presença da fusão TMPRSS2/ERG, e as amostras positivas sequenciadas para determinação do tipo de isoforma envolvida. Identificamos que 35% das amostras continham o rearranjo e que todas correspondiam à isoforma do tipo III, cuja literatura a relaciona com um fenótipo agressivo do CaP, porém não metástico, e é também a mais comumente identificada. Ao confrontarmos essa evidência com os dados clínicos e histopatológicos, constatamos que havia correlação entre eles, sugerindo assim, como em outros trabalhos, o potencial desse rearranjo como marcador de agressividade do CaP. No entanto, não verificamos relação entre a presença da fusão e dados de progressão da doença. Em vista desses resultados, destacamos a necessidade da promoção de outros trabalhos de mesmo caráter, abrangendo outras regiões, a fim de se delinear um perfil mais representativo desse rearranjo no Brasil, uma vez que seu potencial como biomarcador diagnóstico e clínico é enorme e pode influenciar sobremaneira no manejo do CaP. / Scientific interest in gene rearrangements associated with cancer progression and etiogenesis relates mainly to the discovery of BCR/ABL fusion in chronic myelogenous leukemia, and since then there has been an evolution in the management of this disease, prompting a series of related studies in other malignancies. These researches resulted in the meeting of the first gene rearrangement in solid tumors, the fusion gene TMPRSS2/ERG involving the promoter region of the gene of serine protease, TMPRSS2, and the gene family of transcription factors ETS, the ERG. It is specific for adenocarcinoma of the prostate, which makes it a strong candidate biomarker and shows already exert a prominent role in the clinical management of prostate cancer (PCa), as is exercised by the BCR/ABL. Its frequency has been shown to be associated with several factors, especially the ethnic origin. Individuals with CaP from different countries have been studied in the frequency of this merger and the result is quite diverse. In Brazil, however, there is no data about this rearrangement, and this paper aims to contribute to the identification of the same frequency and its contribution to the diagnosis and treatment of PCa in the country. Therefore, we used mRNA from 20 individuals with CaP from the answering service HCFMRP/USP, and by RT-PCR, cDNA obtained from the same people who were investigated for the presence of fusion TMPRSS2/ERG, and positive samples sequenced to determine the type of isoform involved. We found that 35% of the samples contained the rearrangement and that all corresponded to the type III isoform, whose literature relates to an aggressive phenotype of PCa, but not metastatic, and is also the most commonly identified. When we compared this evidence with clinical and histopathological data, we found that there was a correlation between them, suggesting, as in other studies, the potential of this rearrangement as a agressivity marker of PCa. However, no significant association between the presence of data fusion and disease progression. In view of these results, we highlight the need to promote other works of the same character, covering other regions, in order to delineate a more representative profile of this rearrangement in Brazil, since its potential as a biomarker and clinical diagnosis is huge and can influence greatly in the management of PCa.

O interesse científico em rearranjos gênicos relacionados com a etiogênese e progressão do câncer relaciona-se, principalmente, à descoberta da fusão BCR/ABL na Leucemia Mieloide Crônica, sendo que desde então, houve uma evolução no manejo dessa doença, instigando uma série de estudos correlatos em outras neoplasias. Essas pesquisas culminaram no encontro do primeiro rearranjo gênico em tumores sólidos, o gene de fusão TMPRSS2/ERG, envolvendo a região promotora do gene da serina protease, o TMPRSS2, e o gene da família de fatores de transcrição ETS, o ERG. Ele é específico de adenocarcinoma da próstata, o que o torna forte candidato a biomarcador e já demonstra exercer papel de destaque no manejo clínico do câncer de próstata (CaP), tal qual o exercido pela fusão BCR/ABL. Sua frequência têm se mostrado associada a diversos fatores, sobretudo à etnia de origem. Indivíduos portadores de CaP oriundos de diversos países já foram estudados quanto à frequência dessa fusão e o resultado é bastante diversificado. No Brasil, entretanto, ainda não há dados a respeito desse rearranjo, e este trabalho visa contribuir para a identificação da frequência da mesma e sua contribuição para o diagnóstico e o tratamento do CaP no país. Para tal, utilizamos mRNA de 20 indivíduos com CaP provenientes do serviço de atendimento do HCFMRP/USP, e por meio da técnica de RT-PCR, obtivemos o cDNA dos mesmos que foram investigados quanto à presença da fusão TMPRSS2/ERG, e as amostras positivas sequenciadas para determinação do tipo de isoforma envolvida. Identificamos que 35% das amostras continham o rearranjo e que todas correspondiam à isoforma do tipo III, cuja literatura a relaciona com um fenótipo agressivo do CaP, porém não metástico, e é também a mais comumente identificada. Ao confrontarmos essa evidência com os dados clínicos e histopatológicos, constatamos que havia correlação entre eles, sugerindo assim, como em outros trabalhos, o potencial desse rearranjo como marcador de agressividade do CaP. No entanto, não verificamos relação entre a presença da fusão e dados de progressão da doença. Em vista desses resultados, destacamos a necessidade da promoção de outros trabalhos de mesmo caráter, abrangendo outras regiões, a fim de se delinear um perfil mais representativo desse rearranjo no Brasil, uma vez que seu potencial como biomarcador diagnóstico e clínico é enorme e pode influenciar sobremaneira no manejo do CaP. / Scientific interest in gene rearrangements associated with cancer progression and etiogenesis relates mainly to the discovery of BCR/ABL fusion in chronic myelogenous leukemia, and since then there has been an evolution in the management of this disease, prompting a series of related studies in other malignancies. These researches resulted in the meeting of the first gene rearrangement in solid tumors, the fusion gene TMPRSS2/ERG involving the promoter region of the gene of serine protease, TMPRSS2, and the gene family of transcription factors ETS, the ERG. It is specific for adenocarcinoma of the prostate, which makes it a strong candidate biomarker and shows already exert a prominent role in the clinical management of prostate cancer (PCa), as is exercised by the BCR/ABL. Its frequency has been shown to be associated with several factors, especially the ethnic origin. Individuals with CaP from different countries have been studied in the frequency of this merger and the result is quite diverse. In Brazil, however, there is no data about this rearrangement, and this paper aims to contribute to the identification of the same frequency and its contribution to the diagnosis and treatment of PCa in the country. Therefore, we used mRNA from 20 individuals with CaP from the answering service HCFMRP/USP, and by RT-PCR, cDNA obtained from the same people who were investigated for the presence of fusion TMPRSS2/ERG, and positive samples sequenced to determine the type of isoform involved. We found that 35% of the samples contained the rearrangement and that all corresponded to the type III isoform, whose literature relates to an aggressive phenotype of PCa, but not metastatic, and is also the most commonly identified. When we compared this evidence with clinical and histopathological data, we found that there was a correlation between them, suggesting, as in other studies, the potential of this rearrangement as a agressivity marker of PCa. However, no significant association between the presence of data fusion and disease progression. In view of these results, we highlight the need to promote other works of the same character, covering other regions, in order to delineate a more representative profile of this rearrangement in Brazil, since its potential as a biomarker and clinical diagnosis is huge and can influence greatly in the management of PCa.

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.