Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include immunoglobulin heavy chain or T-cell receptor gene rearrangements, recurrent cytogenetic abnormalities and mutations in important hematological genes. Whereas in the majority of adult acute lymphoblastic leukemia patients a suitable MRD target can be identified, in adult acute myeloid leukemia patients well-characterized targets are found in only half of cases. The identification of new specific molecular markers of leukemic blasts for MRD assessment, particularly in AML patients, is therefore highly desirable. Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker. Using a combination of cytogenetic (chromosome banding, chromosome microdissection), molecular cytogenetic (mFISH, mBAND) and molecular biological (next- generation sequencing, long-range...

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.

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include immunoglobulin heavy chain or T-cell receptor gene rearrangements, recurrent cytogenetic abnormalities and mutations in important hematological genes. Whereas in the majority of adult acute lymphoblastic leukemia patients a suitable MRD target can be identified, in adult acute myeloid leukemia patients well-characterized targets are found in only half of cases. The identification of new specific molecular markers of leukemic blasts for MRD assessment, particularly in AML patients, is therefore highly desirable. Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker. Using a combination of cytogenetic (chromosome banding, chromosome microdissection), molecular cytogenetic (mFISH, mBAND) and molecular biological (next- generation sequencing, long-range...

Malignant lymphomas are tumors arising by clonal proliferation of lymphocytes stopped at a specific stage of differentiation. All tumor cells arising from the original clone thus share the same characteristics and that can be used in their detection. Finding a suitable molecular marker of tumor cells is an essential step not only to disease diagnosis, but also for monitoring of minimal residual disease. Minimal residual disease is defined as the subclinical disease level, which malignant cells are not detectable for conventional cytological methods during the therapy. These residual cells can cause relapse. The main goals of the diploma thesis are a detection and analysis of immunoglobulin genes rearrangement and chromosomal translocation t(11; 14) in the MTC region, and a development and optimization of RQ-PCR system for detection of minimal residual disease. Quantification of clonal rearrangement or chromosomal translocation allows the detection of minimal residual disease level in patients with malignant lymphomas. Clonal immunoglobulin genes rearrangement or characteristic chromosomal translocation were analyzed in 19 patients with malignant lymphomas. There were analyzed individual gene segments, N-region and combination variability in immunoglobulin genes rearrangement. There was developed...

Philadelphia (Ph) chromosome, as a result of reciprocal translocation, is in majority of cases connected to two types of leukaemia - chronic myelogenous (CML) and acute lymphoblastic (ALL). The translocation occurs within large intronic sequences of BCR and ABL genes. The breakpoints are specific for individual patient and may be used as a target for monitoring of leukemic burden (MRD, minimal residual disease) during the treatment. In general, MRD is an important prognostic factor, which influences the treatment intensity. Two standardized methods are currently used for its monitoring. The first one is based on the detection of clonal specific Immunoglobulin and/or T-cell receptor genes rearrangements (and thus cannot be used for CML cases) at the DNA level, the second one utilizes detection of the BCR/ABL fusion gene at the mRNA level. Our aim was to optimize and standardize the process to find individual patient breakpoints on Ph chromosome and to use it for MRD quantification. We found the breakpoint in 80 % cases. The MRD data from 15 patients obtained by our method were compared to the levels obtained by standard methods (Ig/TCR and BCR/ABL transcript quantification). In all but 1 patient we found significant discrepancies, raising the questions about leukemic origin and the most accurate method for...

61 Summary Wilms' tumor gene 1 (WT1) is highly expressed in acute leukemia and other hematological malignancies. It has been therefore suggested as a potential universal marker of minimal residual disease (MRD), particularly in patients with acute myeloid leukemia (AML). Due to controversial results of some of the studies, the role of WT1 in MRD follow-up and WT1 prognostic significance remain unclear. WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, despite the increasing number of studies, the clinical impact of WT1 and its isoforms in acute leukemia have not yet been elucidated. We established a unique qPCR method to assess the expression pattern of the main 4 WT1 isoforms. Using this method, we determined the ratio of WT1 variants in the samples of patients with AML, myelodysplastic syndrome (MDS) and healthy controls. Our data showed that this pattern can distinguish among particular hematological malignancies, but lacks a prognostic significance. Within our international study group we determined the prognostic significance of total WT1 expression in childhood AML. Based on our results of a large cohort of patients we can conclude that WT1 expression at...

Analysis of donor chimerism has become a routine method for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). In recent years several groups have also focused on the application of this technique for the detection of relapsing disease after allogeneic HSCT. This review addresses technical issues (sensitivity, specificity) and discusses the advantages and limitations of methods currently used for chimerism analysis and their usefulness for the detection of MRD. In addition, the potential impact of novel procedures, e.g. subset chimerism or real-time PCR-based procedures, is discussed. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Great progress has been achieved in the diagnostics and therapy of childhood acute lymphoblastic leukemia (ALL) during the last few decades and the permanent cure rate for children and adolescents has risen to nearly 90%. The basic principle of ALL treatment is to split patients into several groups receiving treatment of different intensity according to exactly defined prognostic features. This is aimed at reducing both the risk of relapse and toxic complications of treatment. The development of new diagnostic methods, especially in the field of molecular genetics and flow cytometry, allowed further improvements in the risk stratification - the minimal residual disease (MRD) has become a crucial prognostic factor in modern treatment protocols for pediatric ALL as a sensitive marker of both response to therapy and subclinical leukemic involvement of various tissues of the organism. Nevertheless, there is still an intensive search for new markers that would enable even more precise characterization of the leukemic clone, and treatment strategies reflecting the biology of leukemic cells are being optimized. The first part of our study describes the monitoring and prognostic impact of MRD in peripheral blood of children with ALL with emphasis on very early time points of treatment. MRD was examined by the...