O STI (imatinib, Glivec) é um inibidor da tirosina quinase BCR-ABL, responsável pela patogênese da leucemia mielóide crônica (LMC). Um total de 110 pacientes com LMC na fase crônica (FC) que falharam ou foram intolerantes ao tratamento com interferon, fase acelerada (FA) e crise blástica (CB) foram tratados com imatinibe entre dezembro de 2000 e setembro de 2003. Resposta hematológica completa e resposta citogenética maior foram observadas em 95,9% e 69,4% respectivamente em pacientes em FC e 93,2% e 36,4% em FA. Apenas 2 pacientes na CB estão vivos. O imatinib foi bem tolerado com altas taxas de resposta. / STI571 (Imatinib, Glevec) is an inhibitor of the Bcr-Abl tyrosine kinase that is central to the pathogenesis of chronic myelogenous leukemia (CML). A total of 110 patients with CML chronic phase (CP) who failed or were intolerant to interferon, accelerated phase (AP) and blastic crisis (BC) were treated with imatinib from December 2000 until September 2003. Complete hematologic response and major cytogenetic response were observed in 95,9% and 69,4% respectively of patients in CP and 93,2% and 36,4% in AP. Only 2 patients are alive in BC. Imatinib is well tolerated with high rates of response

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 18, 2009). Includes bibliographical references.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T16:15:41Z (GMT). No. of bitstreams: 1 Alberto_FernandoLopes_D.pdf: 5633645 bytes, checksum: 7354434461195623d47c6efe24126229 (MD5) Previous issue date: 2003 / Resumo: Câncer é o resultado de anomalias no genoma acumuladas ao longo do tempo e corresponde a doença genética mais freqüente na população humana. Os últimos anos têm presenciado enormes avanços tecnológicos que facilitaram o desenvolvimento de técnicas para análise em larga escala do conjunto dos genes expressos nos diferentes tecidos, sendo responsável pela explosão do conhecimento em medicina molecular. No presente trabalho avaliamos o transcriptoma da leucemia mielóide crônica através da técnica de ORESTES (Open Reading Frame Expressed Sequence Tags). As seqüências geradas foram agrupadas de acordo com sua homologia através do pacote de bioinformática Phrap. Essa estratégia aumentou o grau de cobertura genômica, já que as seqüências originais apresentavam tamanho máximo de 500 a 700 bases. Após a retirada dos contaminantes, obtivemos 2933 agrupamentos de seqüências (contigs) e 11568 seqüências isoladas. Cada contig representa um gene em potencial e o seu conjunto foi submetido a comparação com o banco de dados público nr para possibilitar a anotação e classificação funcional. Os agrupamentos ORESTES foram classificados em comunicação celular (19%); crescimento, desenvolvimento e manutenção celular (9%); metabolismo celular (30%); estrutura, motilidade e transporte celular (15%); função desconhecida (28%). Adicionalmente, estabelecemos o mapeamento cromossômico dos contigs obtidos, observando distribuição relativamente homogênea e proporcional representada em todos os cromossomos humanos. O mapeamento dos contigs gerados por ORESTES apresentou-se com padrão de distribuição semelhante ao observado por Venter e colaborasores, 2001. Comparamos o padrão de expressão obtido com o transcriptoma de medula óssea de paciente portador de anemia hemolítica hereditária (doença hematológica não neoplásica) gerado por SAGE (Serial Analysis of Gene Expression). Os genes encontrados foram ordenados de acordo com sua representatividade ou nível de expressão. Não houve correlação entre os padrões de expressão observados pos SAGE e por ORESTES. Do conjunto dos 100 genes mais representados nos ORESTES, dez foram selecionados para validação da expressão por PCR quantitativo em tempo real, utilizando o agente fluorescente SybrGreen I. Identificamos e confirmamos o aumento da expressão em nove genes na LMC. Destes, sete tinham aumento superior a 10 vezes o controle (CAMP, ABR, DEFA3, CSF3R, MLL2, S100A8 e MS4A3), com dois deles (DEFA3 e ABR) com aumento de cerca de 50 vezes ou mais. A análise funcional particularizada para cada um deste genes em LMC tem o potencial de identicar possíveis alvos para novas intervenções terapeuticas. O método mostrou-se poderoso para evidenciar e validar as diferenças de expressão gênica observadas pelas técnicas de seqüenciamento utilizadas anteriormente. Os dados deste trabalho constituem a primeira descrição do transcriptoma da leucemia mielóide crônica / Abstract: The complete collection of transcripts generated from the human genome cannot be predicted from the genome sequence, but should be directly determined for each tissue, due to variations of gene expression in different tissues and disease states, and because genes can encode multiple transcripts derived from alternate splicing and polyadenylation sites. As part of larger project that produced up to 1.2 million ORESTES (open reading frames expressed sequence tags) from different cancer tissues, we constructed a set of cDNAs obtained from bone marrow cells of patients with CML, that represented partial expressed gene sequences that are biased toward the central coding regions of the resulting transcripts (Dias-Neto E et al, Proc Nat Acad Sci USA 97:3491, 2000). The 27,361 ESTs were assembled into contigs using the Phrap package producing 2933 clusters containing 2 to 210 ESTs (leaving 11568 isolated sequences). Functional categories were attributed to the contigs on the basis of the annotation of the matched sequences in nr, into 5 major functional categories and 22 subcategories (cell communication (19%); cell growth, development and m contigs formed by the larger number of EST in bone marrow cells was co mpared with a control bone marrow sample obtained from a non-neoplastic hematologic condition (hereditary hemolytic anemia) and evaluated with SAGE (serial analysis of gene expression). Ten out of 100 most representative contigs had their expression data evaluated further by realtime quantitative PCR with Sybr Green I reagent, confirming the ORESTES data. Seven out of the 10 genes were more than 10-fold upregulated (CAMP, ABR, DEFA3, CSF3R, MLL2, S100A8 e MS4A3) and two additional genes were 50-fold or higher upregulated in CML (DEFA3 e ABR). The wealthy of information provided by this approach demonstrates its usefulness for the analysis of gene expression in specific hematopoietic tissues and diseases / Doutorado / Clinica Medica / Doutor em Clínica Médica

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

<p>UVOD: Akutna mijeloidna leukemija (AML) predstavlja heterogenu grupu oboljenja u odnosu na morfologiju, citogenetiku, molekularnu genetiku, zbog čega se deli na različite kliničke i biolo&scaron;ke entitete, sa različitim odgovorom na terapiju i ishodom lečenja. Humani EVI1 (ecotropic virus integration-1) gen ima ulogu multifunkcionalnog nuklearnog transkripcionog faktora, kako u normalnoj tako i u malignoj hematopoezi. Sve je vi&scaron;e istraživanja koja ističu negativni prognostički značaj visoke ekspresije (overexpression) EVI1 gena u AML.&nbsp; CILJEVI: Ciljevi ovog istraživanja su da se ispita klinički i prognostički značaj ekspresije gena EVI1 u AML, kao i da se utvrdi povezanost visoke ekspresije gena EVI1 sa nalazima citogenetskog ispitivanja i molekularnim markerima: FLT3 mutacijom i nukleofozmin 1 (NPM1) mutacijom. MATERIJAL I METODE: Ovim prospektivnim istraživanjem je obuhvaćena grupa od 38 odraslih novodijagnostikovanih bolesnika sa de novo, non M3 AML, kod kojih je započeto standardno lečenje, a koji su dijagnostikovani i lečeni u Klinici za hematologiju Kliničkog centra Vojvodine u periodu od jula 2012. do marta 2014. Određivanje ekspresije gena EVI1 je vr&scaron;eno pomoću real time kvantitativne PCR (qPCR) metode, tehnikom TaqMan, a relativna ekspresija EVI1 gena je određena primenom &Delta;&Delta;Ct metode.&nbsp; REZULTATI: Medijana starosti bolesnika pri postavljanju dijagnoze AML je bila 52 godine (23-80). Ustanovljena je statistički značajna razlika između ekspresije gena EVI1 kod zdravih osoba (kontrolna grupa) i obolelih od akutne mijeloidne leukemije (p=0.008). Računajući relativnu ekspresiju, 13,2 % bolesnika je imalo visoku ekspresiju (overexpression) gena EVI1. U odnosu na kliničke i laboratorijske karakteristike bolesnika (kao &scaron;to su pol, starost, parametri krvne slike, nivo laktat dehidrogenaze, procenat blasta u perifernoj krvi i ko&scaron;tanoj srži, potom tip akutne mijeloidne leukemije, performans status, komorbiditetni indeks) nije ustanovljena statistički značajna razlika između bolesnika sa visokom ekspresijom EVI1 gena i ostalih bolesnika. Postoji statistički značajna povezanost visoke ekspresije EVI1 gena i nepostojanja NPM1 mutacije (p=0,031), kao i između visoke ekspresije EVI1 gena i prisustva monozomije 7 (p=0,047). Visoka ekspresija EVI1 gena je povezana sa kraćim preživaljvanjem bez dogaĎaja (p=0,004), kao i sa kraćim ukupnim preživljavanjem (p=0,025).&nbsp; ZAKLJUČCI: Postoji značajno povećana ekspresija gena EVI1 kod obolelih od AML u odnosu na zdrave kontrole. Visoka ekspresija EVI1 gena je faktor lo&scaron;e prognoze kod obolelih od akutne mijeloidne leukemije i u kombinaciji sa drugim prognostičkim markerima, doprinosi boljoj risk stratifikaciji ovih bolesnika.</p> / <p>INTRODUCTION: Acute myeloid leukaemia (AML) represents a heterogenous group of diseases in terms of morphology, cytogenetics, molecular genetics, so it can be divided into distinct clinical and biological entities, with variable responsiveness to therapy and different treatment outcome. Human EVI1 (ecotropic virus integration-1) gene plays a role of multifunctional nuclear transcriptional factor, not only in normal, but also in malignant haematopoiesis. There are more and more investigations indicating high EVI1 expression (EVI1 overexpression) as a negative prognostic marker in AML.&nbsp; PURPOSES: The main goal of this investigation was to examine the clinical and prognostic significance of EVI1 expression in AML, as well as to investigate whether there was any association of EVI1 overexpression with cytogenetic abnormalities and other standard molecular prognostic factors, such as FLT3 mutation and nucleophosmin 1 (NPM1) mutation.&nbsp; PATIENTS AND METHODS: This prospective study included 38 adult newly diagnosed patients with de novo nonM3 AML, in whom a standard treatment was started at Clinic of Haematology, Clinical center of Vojvodina in the period from July 2012 to March 2014. EVI1 expression was analyzed by real-time quantitative polymerase chain reaction using TaqMan, and relative EVI1 expression was determined by &Delta;&Delta;Ct method.&nbsp; RESULTS: Median age of patients at diagnosis was 52 (aged 23-80). There has been determined statistically higher EVI1 expression in our AML patients than in healthy volunteers (control group) (p=0.008). The relative EVI1 overexpression was observed in 13.2% of the patients. No significant differences in clinical and laboratory patient data (including sex, age, whole blood counts, lactate dehydrogenase level, peripheral and bone marrow blast percentages, type of AML, performance status, comorbidity index) were observed between patients with high EVI1 expression and patients without high EVI1 expression. Our investigation revealed inverse correlation of high EVI1 expression and nucleophosmin 1 mutation (p=0,031). Also high EVI1 expression was significantly associated with monosomy 7 (p=0,047). Survival analysis revealed significantly inferior event free survival (p=0,004) and overall survival (p=0,025) for patients with high EVI1 expression compared to the other patients.&nbsp; CONCLUSION: EVI1 expression is significantly higher in AML patients compared to healthy controls. High EVI1 expression is a poor prognostic marker for patients with AML, and in combination with other well established prognostic markers, contributes to better risk stratification of these patients.</p>

Foram analisados os resultados retrospectivos do transplante de medula óssea alogênico em 100 pacientes portadores de leucemia mielóide crônica, divididos em dois grupos: Grupo I, de 49 pacientes que recebeu alta hospitalar após a pega medular, e grupo II, de 51 pacientes, que recebeu alta precocemente, antes da pega medular, para acompanhamento ambulatorial. Foram comparados os dias de ocupação de leitos hospitalares, a morbidade e a mortalidade entre os grupos de alta até o dia 100 pós transplante. Verificou-se que, no grupo de alta precoce, houve, significativamente, menos dias de ocupação de leitos hospitalares, sem aumento da morbidade e da mortalidade / We analyzed the results of a retrospective study of 100 patients with chronic myelogenous leukemia submitted to allogeneic stem cell transplantation in one of two settings: Group I, with 49 patients, transplanted in the traditional inpatient and group II, with 51 patients, in partial outpatient. We compared the median number of days spent in hospital, morbidity and mortality within 100 after bone marrow transplantation. We concluded that there was a significant reduction in the median of hospital length of stay in the partial outpatient group, without increasing morbidity and mortality

Foram analisados os resultados retrospectivos do transplante de medula óssea alogênico em 100 pacientes portadores de leucemia mielóide crônica, divididos em dois grupos: Grupo I, de 49 pacientes que recebeu alta hospitalar após a pega medular, e grupo II, de 51 pacientes, que recebeu alta precocemente, antes da pega medular, para acompanhamento ambulatorial. Foram comparados os dias de ocupação de leitos hospitalares, a morbidade e a mortalidade entre os grupos de alta até o dia 100 pós transplante. Verificou-se que, no grupo de alta precoce, houve, significativamente, menos dias de ocupação de leitos hospitalares, sem aumento da morbidade e da mortalidade / We analyzed the results of a retrospective study of 100 patients with chronic myelogenous leukemia submitted to allogeneic stem cell transplantation in one of two settings: Group I, with 49 patients, transplanted in the traditional inpatient and group II, with 51 patients, in partial outpatient. We compared the median number of days spent in hospital, morbidity and mortality within 100 after bone marrow transplantation. We concluded that there was a significant reduction in the median of hospital length of stay in the partial outpatient group, without increasing morbidity and mortality