Atualmente, a Leucemia Linfocítica Crônica (LLC) pode ser dividida em dois grupos: um com mutações somáticas no gene da região variável da cadeia pesada da imunoglobulina (MIgVH) e outro sem mutações (NMIgVH). Alguns estudos mostraram que a expressão de CD38 na superfície das células B de LLC pode estar correlacionada com o estado mutacional do gene VHIg, entretanto, esses controversos. Estudos recentes mostraram que a expressão da proteína tirosina quinase Zap-70 está melhor associada com o estado mutacional do gene IgVH. O objetivo deste estudo foi avaliar a expressão de Zap-70 e CD38, por citometria de fluxo, nas células CD19+ de pacientes com LLC e correlacioná-los com o estádio clínico (EC), sobrevida livre de tratamento (SLT) e sobrevida global (SG). A expressão de Zap-70 e CD38 foi avaliada, em 144 de pacientes com LLC classificados nos estádios clínicos A, B e C de acordo com os critérios de Binet: 59 (41%) do EC-A, 38 (26%) do EC-B e 47 (33%) do EC-C. Foi observada menor positividade para Zap-70 e CD38 nos pacientes do EC-A do que nos EC-B e C. Quando avaliada a SLT nos pacientes do EC-A, os casos Zap-70+ assim como os CD38+ apresentaram menor SLT. A média de SG dos pacientes Zap-70+ e CD38+ foi menor quando comparado com os Zap-70- e CD38- entretanto quando correlacionada com o EC não foi observada diferença estatisticamente significante entre a expressão desses marcadores e o EC-A, B ou C. Pela analise combinada de CD38 e Zap-70, dividimos os pacientes em dois grupos (Zap-70-/CD38- e Zap-70+ ou CD38+). Observamos que a expressão positiva desses dois marcadores estava associada ao EC, uma vez que a grande maioria dos pacientes dos estádios B (74%) e C (66%) expressam Zap-70 ou CD38. Entretanto, os pacientes do EC-A, Zap-70+ ou CD38+, apresentaram SG menor quando comparado com os Zap-70-/CD38-. Essa diferença não foi observada nos pacientes do EC-B e do EC-C. Também foi observada menor SLT nos pacientes no EC-A, Zap-70+ ou CD38+. Esses resultados sugerem que análise combinada de Zap-70 e CD38 podem ser empregadas na avaliação dos pacientes do EC-A para se acompanhar a evolução clinica desse grupo de pacientes. Porém, estudos adicionais devem ser realizados para se validar a utilização clínica desses marcadores. / Actually, chronic lymphocytic leukemia (CLL) can be divided in two subsets: one with somatically mutated immunoglobulin heavy-chain variable-region genes (MIgVH) and other with unmutated sequences. (UMIgVH). Some studies have shown that CD38 expression in CLL cells are correlated with IgVH mutational status. However, the value of CD38 as surrogate IgVH mutational status is controversial. Recent studies, have found that Zap-70 protein tyrosine kinase expression is strongly associated with the mutational status IgVH. The aim of this study was to evaluate the Zap-70 and CD38 expression, for flow cytometry, in CD19+ LLC cells and correlate with the Binet’s staging system, treatment-free survival (TFS) and a overall survival (OS). Zap-70 and CD38 was evaluated, in 144 CLL patients that was classified in A, B and C Binet’s staging system: 59 (41%) in stage A, 38 (26%) in B and 47 (33%) in C. We observed low Zap-70 and CD38 expression in stage A patients than in stage B and C cases. When we analyzed the TFS in stage A patients Zap-70+ and CD38+ patients showed shorter TFS than Zap-70- and CD38-. Then we observed that the OS of Zap-70+ and CD38+ patients was, also, shorter than Zap-70- and CD38- cases. However, statistical differences was not found when Zap-70 and CD38 expression was correlated with stage A, B or C Binet’s staging system. To understand the associated Zap-70 and CD38 expression, we divided the CLL patients in two subgroups (Zap-70-/CD38 - and Zap-70+ or CD38+). We observed that CD38+ or Zap-70+ was associated Binet’s staging system, once most of stage B (74%) and C (66%) patients are Zap-70+ or CD38+. However, stage A patients, Zap-70+ or CD38+, showed shorter OS than Zap-70-/CD38-. These differences were not observed in stage B and C patients. Shorter TFS was also observed in the Zap-70+ or CD38+ stage A patients. These results suggest that combined analysis of Zap-70 and CD38 can be used to evaluate stage A patients to observe the clinical evolution of the disease. Nevertheless, other studies must be carried to confirm the clinical use of these markers.

The presence of TP53 mutations has been associated with inferior outcome in diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In DLBCL, the impact of the TP53 codon 72 polymorphism and MDM2 SNP309 has not been clearly elucidated, whereas MDM2 SNP309 was suggested as a poor-prognostic marker in CLL. In addition, p16INK4a promoter hypermethylation has been implicated as a negative prognostic factor in DLBCL. The aim of this thesis was to further evaluate these molecular markers in well-characterised materials of DLBCL and CLL. In paper I, we investigated the prognostic role of TP53 mutation, codon 72 polymorphism and MDM2 SNP309 in DLBCL (n=102). The presence of TP53 mutations (12.7%) correlated with a poor lymphoma-specific and progression-free survival, and a particularly pronounced effect was observed in the germinal center subtype. Neither the MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. In paper II, we applied pyrosequencing to measure the level of p16INK4a methylation in DLBCL (n=113). Thirty-seven percent of cases displayed p16INK4a methylation; however, no clear association could be observed between degree of methylation and clinical characteristics or lymphoma-specific survival. In papers III–IV, we investigated the prognostic role of MDM2 SNP309 (n=418) and TP53 mutation (n=268) in CLL. No correlation was observed between any particular MDM2 SNP309 genotype and time to treatment and overall survival. Furthermore, no association was found between the different MDM2 SNP309 genotypes and established CLL prognostic markers. TP53 mutations were detected in 3.7% of CLL patients; where the majority showed a concomitant 17p-deletion and only three carried TP53 mutations without 17p-deletion. We confirmed a significantly shorter overall survival and time to treatment in patients with both TP53 mutation and 17p-deletion. Altogether, our studies could confirm the negative prognostic impact of TP53 mutations in DLBCL, whereas MDM2 SNP309 and TP53 codon 72 polymorphisms appear to lack clinical relevance. We also question the role of p16INKa methylation as a poor-prognostic factor in DLBCL. Finally, the presence of TP53 mutation in CLL appears to be rare at disease onset and instead arise during disease progression.

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy displaying varying clinical outcome, where molecular markers today can divide patients into prognostic subgroups. Despite the introduction of new agents for treatment, remissions are usually not sustained in CLL and resistance towards treatment can partly be explained by aberrant apoptosis. The aim of this thesis was to find new drugs for CLL patients resistant to conventional therapy and to analyze genes involved in apoptosis within different prognostic subgroups. In paper I-II, the in vitro activity of substances was investigated using the fluorometric microculture cytotoxicity assay (FMCA). When evaluating rapamycin (paper I), an inhibitor of mTOR, in 97 tumor samples from different entities, CLL was found to be one of the most sensitive tumor types. Combination experiments on patient CLL cells indicated that rapamycin acted synergistically with the CLL drugs vincristine and chlorambucil. An investigation of 20 anti-cancer agents in cells from 40 CLL patients (paper II) revealed that prednisolone and rolipram displayed high activity in poor-prognostic patients, in particular IGHV unmutated CLL. Furthermore, when used in combination these agents were found to produce a synergistic effect. In paper III, the anti-apoptotic BCL2 family member BFL1 was evaluated in 37 CLL cases. Levels of BFL1 were higher in fludarabine-resistant patients compared to fludarabine-sensitive patients. In addition, the high expression of BFL1 inversely correlated to fludarabine-induced apoptosis in CLL cells. A single nucleotide polymorphism in the anti-apoptotic BCL2 gene (-938C>A) has been suggested as a novel poor-prognostic marker in CLL. In paper IV, we investigated this BCL2 polymorphism in 268 CLL patients and correlated genotypes to clinical data. However, no association could be confirmed between this polymorphism and clinical outcome or established prognostic markers. In conclusion, this thesis has shown that rapamycin is a potential drug for treatment in CLL. Furthermore, prednisolone and rolipram were identified as interesting candidates for treatment of poor-prognostic patients. Finally, the anti-apoptotic protein BFL1 may contribute to chemoresistance and hence represents a potential therapeutic target in CLL, whereas from our data, the BCL2 -938C>A polymorphism does not appear to have any prognostic significance.

Η παρούσα διδακτορική διατριβή έχει ως στόχο τη δημιουργία ενός αποδοτικού μοντέλου για το Λειτουργικό Συνδυασμό Βιο-Ιατρικών δεδομένων (BioMedical data integration). Ξεκινώντας από τη σχεδιαστική ανάλυση της ιατρικής γνώσης και των προβλημάτων που προκύπτουν από τον τρόπο παραγωγής των ιατρικών δεδομένων, προχωρεί στην επίλυση των επιμέρους θεμάτων Λειτουργικού Συνδυασμού εντός ενός συγκεκριμένου ιατρικού πεδίου και καταλήγει στον ολοκληρωμένο Λειτουργικό Συνδυασμό ιατρικών δεδομένων προερχόμενων από διαφορετικές πηγές και πεδία γνώσης. Συνεχίζει με τη σχεδίαση ενός μοντέλου βάσεων δεδομένων που ακολουθεί «οριζόντια» λογική και είναι αρκετά αποδοτικό ώστε να αποκρίνεται σε πολύπλοκα και ευρείας κλίμακας ερωτήματα σε πραγματικό χρόνο. Καταλήγει με την παρουσίαση μίας ολοκληρωμένης εφαρμογής η οποία εκμεταλλευόμενη τα πλεονεκτήματα του Λειτουργικού Συνδυασμού και της οριζόντιας δομής των δεδομένων είναι σε θέση να διαχειριστεί εξετάσεις προερχόμενες από κάθε κυτταρομετρητή ροής και συνδυάζοντάς αυτές με τις υπόλοιπες αιματολογικές κλινικοεργαστηριακές εξετάσεις να απαντά σε καθημερινά και σύνθετα ερευνητικά, ιατρικά ερωτήματα. Τα πρωτότυπα ερευνητικά αποτελέσματα που προέκυψαν στα πλαίσια της παρούσης εργασίας δημοσιεύτηκαν σε έγκυρα διεθνή περιοδικά και σε διεθνή και ελληνικά συνέδρια με κριτές. / Current dissertation focuses on the creation of an efficient model for Bio-medical data integration. Starting with an analytical approach of the medical knowledge and the problems that may occur cause of the way that medical data are produced, continues with the necessary solutions for single domain data integration and concludes with the proposal of a working framework for mass data integration, originating from multiple medical domains. The proposed integration model is based on the “horizontal” logic of a database design and it’s efficient enough to produce query results in real time, even for complex real-life medical questions. The proof of concept of the working framework and its goals for mass data integration is achieved through the presentation of a medical information system. The presented system, by taking advantage of the “horizontal” database design, is able to manage Flow Cytometry measurements, originating for any available hardware and by integrating the cytometric data with other types of hematological data is able to give answers to everyday and research medical questions. All original research results that produced within the scope of this dissertation were published in international research journals and medical conferences.

The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, which has prompted the search for biomarkers that can predict prognosis in this disease. The IGHV gene mutation status and certain genomic aberrations have been identified as reliable prognostic markers of clinical outcome for this disorder. However, the search for more feasible prognostic markers in CLL is still being pursued. Recently, certain single nucleotide polymorphisms (SNPs) in the GNAS1, BCL2 and MDM2 genes and the RNA expression levels of the LPL, ZAP70, TCL1, CLLU1 and MCL1 genes were suggested as novel prognostic markers in CLL. In papers I-III, we performed genotyping analyses of the GNAS1 T393C, BCL2 -938C>A and MDM2 SNP309 polymorphisms in 268-418 CLL patients and related the genotypes with clinical data. Association studies between the polymorphisms and established prognostic markers (i.e. IGHV mutation status, genomic aberrations, CD38 expression) were also performed. Our studies did not find any significant relationship between these SNPs with either clinical outcome or other known prognostic markers in CLL. In paper IV, we measured the RNA expression levels of LPL, ZAP70, TCL1, CLLU1 and MCL1 in 252 CLL cases and correlated these levels with clinical outcome. Here, we verified that high expression of all these RNA-based markers, except MCL1, were associated with an unfavourable prognosis. We also confirmed a close relationship between IGHV mutation status and the RNA-based markers, especially for LPL and CLLU1 expression. Among the RNA-based markers, multivariate analysis revealed LPL expression as the strongest independent prognostic marker for overall survival and time to treatment. Furthermore, the RNA-based markers could add further prognostic information to established markers in subgroups of patients, with LPL expression status giving the most significant results. In summary, data from papers I-III could not verify the GNAS1 T393C, BCL2 -938C>A and MDM2 SNP309 polymorphisms as prognostic markers in CLL. Future SNP markers must hence be confirmed in large, independent cohorts before being proposed as prognostic marker in CLL. In paper IV, we conclude that LPL expression appears to be the strongest among the RNA-based markers for CLL prognostication. Further efforts to standardize LPL quantification are required before it can be applied in the clinical laboratory to predict clinical outcome in this disease.

Η Β-χρόνια Λεμφογενής Λευχαιμία (Β-ΧΛΛ) αποτελεί τον πιο κοινό τύπο λευχαιμίας στο Δυτικό κόσμο. Η πρόγνωσή της θεωρείται ως ένα από τα πιο ενδιαφέροντα προβλήματα απόφασης στην κλινική έρευνα και πρακτική. Για διάφορους κλινικούς και εργαστηριακούς δείκτες είναι γνωστό ότι σχετίζονται με την εξέλιξη της νόσου. Για τις παραμέτρους, όμως, που εξάγονται με ανάλυση κυτταρομετρίας ροής, οι οποίες αποτελούν τον ακρογωνιαίο λίθο της διαδικασίας διάγνωσης της νόσου, το αν προσφέρουν επιπρόσθετη προγνωστική πληροφορία αποτελεί ανοιχτό πρόβλημα. Στη διατριβή αυτή προτείνουμε ένα σύστημα υποβοήθησης για τις αποφάσεις των ειδικών του πεδίου, το οποίο πραγματοποιεί πολυπαραμετρική πρόγνωση ασθενών με Β-ΧΛΛ, συνδυάζοντας τη χρήση ποικίλων ετερογενών προγνωστικών δεικτών (κλινικών, εργαστηριακών και κυτταρομετρίας ροής) που σχετίζονται με τη νόσο. Η διάγνωση της Β-ΧΛΛ βασίζεται κυρίως στη μελέτη του αντιγονικού φαινότυπου των κυττάρων των ασθενών, η οποία διενεργείται με κυτταρομετρία ροής. Αν και η διαδικασία που ακολουθείται κατά την ανάλυση αυτή είναι σαφώς ορισμένη, ο τρόπος με τον οποίο οι εργαστηριακοί υπεύθυνοι την πραγματοποιούν παραδοσιακά χαρακτηρίζεται από ανακρίβεια και υποκειμενικότητα. Καθώς η τεχνολογία της κυτταρομετρίας ροής εξελίσσεται ραγδαία, γίνεται όλο και πιο επιτακτική η ανάγκη για την ανάπτυξη αυτοματοποιημένων μεθόδων ανάλυσης των δεδομένων που παράγει. Σε αυτά τα πλαίσια, παρουσιάζουμε ένα χρήσιμο παράδειγμα αυτοματοποιημένης ανάλυσης κυτταρομετρικών δεδομένων, η οποία δεν απαιτεί την άμεση επίβλεψη των ειδικών, για τη διάγνωση ασθενών με Β-ΧΛΛ. Οι τιμές των χαρακτηριστικών παραμέτρων που εξάγονται με εφαρμογή της προτεινόμενης μεθοδολογίας, ενσωματώνονται κατόπιν στο προαναφερθέν προγνωστικό σύστημα. Ανάγοντας το πρόβλημα της πρόγνωσης της Β-ΧΛΛ σε ένα στιγμιότυπο ταξινόμησης προτύπων, καθώς και προσομοιώνοντας κάθε ένα από τα βήματα της διαδικασίας της διάγνωσης της νόσου με ένα στιγμιότυπο συσταδοποίησης δεδομένων, αντιμετωπίσαμε τα δύο προβλήματα εφαρμόζοντας τεχνικές στατιστικής μάθησης. Εστιάσαμε σε μεθοδολογίες δικτύων πεποίθησης, χρησιμοποιώντας συγκεκριμένα το naïve-Bayes μοντέλο και για τις δύο περιπτώσεις, στην επιβλεπόμενη και στη μη επιβλεπόμενη εκδοχή του, αντίστοιχα. Τα χαρακτηριστικά και η φύση των δεδομένων (κυρίως των κυτταρομετρικών) που παράγονται από έναν παθολογικό υποκείμενο μηχανισμό, όπως αυτός της νόσου, δεν ευνοούν την απευθείας εφαρμογή του παραπάνω μοντέλου στο εκάστοτε στιγμιότυπο. Για το λόγο αυτό, συνδυάσαμε την εφαρμογή του naïve-Bayes μοντέλου με κατάλληλες ευρετικές αλγοριθμικές διαδικασίες, για την επίτευξη καλύτερων αποτελεσμάτων, με κριτήριο βέλτιστου όχι μόνο κάποιες συχνά χρησιμοποιούμενες μετρικές αποτίμησης αλγόριθμων, αλλά και τη γνώμη των αιματολόγων. Χάρη στην ιδιότητά τους να ενσωματώνουν την έμπειρη γνώση των ειδικών ως εκ των προτέρων πληροφορία αρχικοποίησης των μεθόδων μάθησής τους, οι Bayesian μεθοδολογίες κρίνονται ως οι πλέον κατάλληλες για την εφαρμογή τους σε τέτοιου τύπου προβλήματα. / B-Chronic Lymphocytic Leukemia (B-CLL) is known to be the most common type of leukemia in the Western world. Its prognosis remains one of the most interesting decision problems in clinical research and practice. Various clinical and laboratory factors are known to be associated with the evolution of the disease. However, for the parameters obtained by flow cytometry analysis, that are traditionally utilized as the cornerstone during the diagnosis procedure of the disease, whether they offer additional prognostic information is an open issue. In this dissertation, we propose a decision support system to the hematologists, that provides multiparametric B-CLL patients’ prognosis, combining the usage of diverse heterogeneous factors (clinical, laboratory and flow cytometry) associated with the disease. B-CLL diagnosis is primarily derived from the study of the antigenic phenotype of the patients’ blood cells, which is held with flow cytometry analysis. Despite the fact that the method of the analysis is well defined, the process traditionally followed by the laboratory experts is characterized by amounts of inexactness and subjectivity. As flow cytometry technology advances rapidly, the need for adequate automated (computer-assisted) analysis methodologies on the data it produces is accordingly increasing. In this context, we present a useful paradigm of automated analysis of flow cytometry data, that does not require the direct supervision of the expert, for B-CLL patients’ diagnosis. The values of the flow cytometry characteristic parameters extracted by applying the proposed methodology are afterward incorporated to the prognostic system for B-CLL mentioned above. By reducing the B-CLL prognosis problem to an instance of the pattern classification problem, as well as by simulating each step of the B-CLL diagnosis procedure with an instance of the data classification problem, we proceeded with applying statistical learning techniques. We focused on Bayesian network methodologies and utilized the naïve-Bayes model for both cases, in its supervised and unsupervised version, respectively. The characteristics of the data (especially of the flow cytometry ones) generated by a pathological underlying mechanism, like the disease’s one, did not encourage the direct use of the above model. Therefore, we combined the naïve-Bayes model with a set of suitable heuristic algorithmic procedures to obtain better results, not only with respect to some commonly used algorithmic optimality metrics, but also by considering the experts’ opinion. Due to their ability of incorporating the expert knowledge as a priori initial information to their learning methods, Bayesian methodologies are considered as the most appropriate ones to make use of in such types of applications.

Chronic Lymphocytic Leukemia is the primary lymphoid neoplasm in adults and and it is especially manifested in the elderly. Because it is a heterogeneous disease it awakens great interest regarding its prognosis. Rai and Binet developed staging systems to predict the evolution of the disease and currently, the analysis of expression of CD38 and Zap-70 has been investigated as a prognostic factor for indicating presence or absence of the mutation in the gene IgVH, so, the objective of this study was to analyze the clinical and laboratory profiles of patients with Chronic Lymphocytic Leukemia taking as reference the clinical staging of Rai and Binet and quantification of CD38 and Zap-70 expression as prognosis factors. We searched the medical records of 64 patients treated at University Hospital of Santa Maria and the variables considered were swollen lymph nodes, presence or absence of hepatomegaly and / or splenomegaly, hematological evaluation of peripheral blood and immunophenotype. The data obtained were correlated with the staging of Rai (1975) and Binet (1981), the expression of CD38 and Zap-70 and clinical stage. The results showed no association between ataging Rai and Binet and the expression of CD38, Zap-70 and Binet clinical staging. / A Leucemia Linfocítica Crônica é a principal neoplasia linfóide em adultos e se manifeta principalmente em indivíduos idosos. Por ser uma doença heterogênea, desperta grande interesse quanto ao seu prognóstico. Rai e Binet desenvolveram sistemas de estadiamento capazes de prever a evolução da doença e atualmente, a análise da expressão de CD38 e Zap- 70 tem sido investigada como fator prognóstico por indicar presença ou ausência da mutação no gene IgVH, assim, o objetivo deste estudo foi analisar o perfil clínico-laboratorial dos pacientes com Leucema Linfocítica Crônica, tomando como referência os estadiamentos clínicos de Rai e Binet e a quantificação da expressão de CD38 e Zap-70 como fatores prognóstico. Foram pesquisados 64 prontuários médicos de pacientes atendidos no Hospital Universitário de Santa Maria e as variáveis consideradas foram aumento de linfonodos, presença ou ausência de hepatomegalia e/ou esplenomegalia, avaliação hematológica de sangue periférico e imunofenótipo. Os dados obtidos foram correlacionados com o estadiamento de Rai (1975) e Binet (1981), a expressão de CD38 e Zap-70 com o estádio clínico de Binet. Os resultados demonstraram que não há associação entre o estadiamento de Raí e Binet e a expressão de CD38, Zap-70 com o estadiamento clínico de Binet.

Atualmente, a Leucemia Linfocítica Crônica (LLC) pode ser dividida em dois grupos: um com mutações somáticas no gene da região variável da cadeia pesada da imunoglobulina (MIgVH) e outro sem mutações (NMIgVH). Alguns estudos mostraram que a expressão de CD38 na superfície das células B de LLC pode estar correlacionada com o estado mutacional do gene VHIg, entretanto, esses controversos. Estudos recentes mostraram que a expressão da proteína tirosina quinase Zap-70 está melhor associada com o estado mutacional do gene IgVH. O objetivo deste estudo foi avaliar a expressão de Zap-70 e CD38, por citometria de fluxo, nas células CD19+ de pacientes com LLC e correlacioná-los com o estádio clínico (EC), sobrevida livre de tratamento (SLT) e sobrevida global (SG). A expressão de Zap-70 e CD38 foi avaliada, em 144 de pacientes com LLC classificados nos estádios clínicos A, B e C de acordo com os critérios de Binet: 59 (41%) do EC-A, 38 (26%) do EC-B e 47 (33%) do EC-C. Foi observada menor positividade para Zap-70 e CD38 nos pacientes do EC-A do que nos EC-B e C. Quando avaliada a SLT nos pacientes do EC-A, os casos Zap-70+ assim como os CD38+ apresentaram menor SLT. A média de SG dos pacientes Zap-70+ e CD38+ foi menor quando comparado com os Zap-70- e CD38- entretanto quando correlacionada com o EC não foi observada diferença estatisticamente significante entre a expressão desses marcadores e o EC-A, B ou C. Pela analise combinada de CD38 e Zap-70, dividimos os pacientes em dois grupos (Zap-70-/CD38- e Zap-70+ ou CD38+). Observamos que a expressão positiva desses dois marcadores estava associada ao EC, uma vez que a grande maioria dos pacientes dos estádios B (74%) e C (66%) expressam Zap-70 ou CD38. Entretanto, os pacientes do EC-A, Zap-70+ ou CD38+, apresentaram SG menor quando comparado com os Zap-70-/CD38-. Essa diferença não foi observada nos pacientes do EC-B e do EC-C. Também foi observada menor SLT nos pacientes no EC-A, Zap-70+ ou CD38+. Esses resultados sugerem que análise combinada de Zap-70 e CD38 podem ser empregadas na avaliação dos pacientes do EC-A para se acompanhar a evolução clinica desse grupo de pacientes. Porém, estudos adicionais devem ser realizados para se validar a utilização clínica desses marcadores. / Actually, chronic lymphocytic leukemia (CLL) can be divided in two subsets: one with somatically mutated immunoglobulin heavy-chain variable-region genes (MIgVH) and other with unmutated sequences. (UMIgVH). Some studies have shown that CD38 expression in CLL cells are correlated with IgVH mutational status. However, the value of CD38 as surrogate IgVH mutational status is controversial. Recent studies, have found that Zap-70 protein tyrosine kinase expression is strongly associated with the mutational status IgVH. The aim of this study was to evaluate the Zap-70 and CD38 expression, for flow cytometry, in CD19+ LLC cells and correlate with the Binet’s staging system, treatment-free survival (TFS) and a overall survival (OS). Zap-70 and CD38 was evaluated, in 144 CLL patients that was classified in A, B and C Binet’s staging system: 59 (41%) in stage A, 38 (26%) in B and 47 (33%) in C. We observed low Zap-70 and CD38 expression in stage A patients than in stage B and C cases. When we analyzed the TFS in stage A patients Zap-70+ and CD38+ patients showed shorter TFS than Zap-70- and CD38-. Then we observed that the OS of Zap-70+ and CD38+ patients was, also, shorter than Zap-70- and CD38- cases. However, statistical differences was not found when Zap-70 and CD38 expression was correlated with stage A, B or C Binet’s staging system. To understand the associated Zap-70 and CD38 expression, we divided the CLL patients in two subgroups (Zap-70-/CD38 - and Zap-70+ or CD38+). We observed that CD38+ or Zap-70+ was associated Binet’s staging system, once most of stage B (74%) and C (66%) patients are Zap-70+ or CD38+. However, stage A patients, Zap-70+ or CD38+, showed shorter OS than Zap-70-/CD38-. These differences were not observed in stage B and C patients. Shorter TFS was also observed in the Zap-70+ or CD38+ stage A patients. These results suggest that combined analysis of Zap-70 and CD38 can be used to evaluate stage A patients to observe the clinical evolution of the disease. Nevertheless, other studies must be carried to confirm the clinical use of these markers.

Genomic integrity is important for living cells' correct functioning and propagation. Deoxyribonucleic acid as a molecule is a subject to chemical reactions with agents that can come from environment as well as from internal metabolism processes. These reactions can induce damage to DNA and thus compromise the genetic information, and result in disease and death of an organism. To mitigate the damage to DNA, cells have evolved to have multiple DNA repair pathways. Presented here is a computational study of DNA repair genes. The structure of the Homo sapiens direct DNA repair gene ALKBH1 is predicted utilizing homology modeling methods and using AlkB and DBL proteins as templates. Analysis of the obtained structure and molecular dynamics simulations give insights into potentially functionally important residues of the protein. In particular, zinc finger domains are predicted, and lysines that could perform catalytic activities are investigated. Subsequent mutagenesis experiments revealed the effect of the residues predicted to form zinc fingers on activity of ALKBH1. Structure and dynamics of AlkD, a Bascillus cereus base excision DNA repair protein is also studied. This protein has been shown to bind DNA with large alkyl adducts and perform excision catalysis without base flipping which is characteristic to other enzymes in the same family. MD simulations of AlkD revealed that B helix, which interacts with DNA, has higher fluctuations when AlkD is not bound to DNA, and thus could have a role in binding and recognition of DNA. For the purpose of finding biomarkers and to further our understanding of a mode of action of DNA repair genes, statistical methods were applied to identify mutations that are linked to cancer phenotypes. Analysis was based on case-control studies of patients with cancers of prostate, breast, pancreas, lung as well as chronic lymphocytic leukemia from NCBI dbGAP database. Those mutations that result in missense mutations were further investigated. In particular, extensive MD simulations and experimental investigations were performed on the mutation in the ALKBH7 gene that was found to be linked to prostate cancer.

Bien que l’utilité de la TEP au [18F]FDG soit confirmée pour le diagnostic et le suivi thérapeutique chez les patients atteints de lymphome, la spécificité de la captation du [18F]FDG a été mise en doute en raison de sa dépendance au métabolisme du glucose, qui peut augmenter dans des conditions bégnines comme les processus inflammatoire ou infectieux. Compte tenu de ces limites, un nucléoside a été développé en tant que nouvel outil pour l'imagerie TEP ([18F]fludarabine). Une radiosynthèse entièrement automatisée a été mise en place et des études précliniques ont été menées sur des modèles murins de xénogreffes (lignées cellulaires folliculaires: RL7 et DOHH-2, myélome multiple (MM) : RPMI8226, lymphome du système nerveux central (LSNC) : MC116), parallèlement au [18F]FDG. Le profil de distribution de la radioactivité de la [18F]fludarabine dans des organes sélectionnés a confirmé le ciblage spécifique de la tumeur. Dans un modèle de lymphome folliculaire, nous avons évalué sa fiabilité pendant le traitement par rituximab et démontré - pas d’interférence entre le traitement et sa captation - une plus forte corrélation entre la fixation de ce radiopharmaceutique et les valeurs quantitatives extraites de l'histologie, comparée au [18F]FDG-TEP. En conséquence, cet outil TEP peut être considéré comme une approche prometteuse pour détecter la maladie résiduelle persistante pendant ou après un traitement par chimiothérapie. En outre, l’évaluation de la [18F]fludarabine lors d’un processus inflammatoire induit par la térébenthine a montré une accumulation significativement plus faible dans le tissu inflammé par comparaison à celle observée avec le [18F]FDG. Dans le MM, le rôle de la [18F]FDG-TEP reste limité en raison de son manque de sensibilité pour détecter une atteinte diffuse de la moelle osseuse et de petites lésions crâniennes dues à la fixation physiologique du [18F]FDG dans le cerveau. Nos données suggèrent que la TEP à la [18F]fludarabine pourrait représenter une modalité alternative et peut-être plus spécifique pour l'imagerie de MM. Dans notre dernière étude, sur les tumeurs cérébrales de xénogreffes, ce nouvel outil TEP a révélé des réponses significativement divergentes entre le LSNC et le glioblastome (GBM), tandis que le [18F]FDG a montré un chevauchement de fixation entre ces deux modèles. Une première étude chez l'homme a été entreprise pour un diagnostic initial, où 10 patients non traités ont été recrutés avec une leucémie lymphoïde chronique à cellules B (LLC) ou un lymphome B diffus à grandes cellules (LBDGC). Les scanners partiels successifs ont été acquis pendant 250 mn après l’injection i.v d’une activité de 4MBq/kg. Les résultats avec les modalités conventionnelles (TDM et/ou [18F]FDG-TEP) ont également été considérés. L'étude a également été conçue pour estimer la dosimétrie pour les principaux organes. Chez les patients atteints d’un LBDGC, une augmentation de la captation a été observée dans les sites considérés anormaux par TDM et [18F]FDG ; chez deux patients, des résultats contradictoires ont été observés avec ces deux radiopharmaceutiques. Chez les patients LLC, la fixation de la [18F]fludarabine a coïncidé avec les sites susceptibles d'être impliqués et a montré une captation significative dans la moelle osseuse hématopoïétique. Aucune fixation n'a été observée, quel que soit le groupe de maladies, dans le muscle cardiaque et le cerveau. De plus, la dose efficace moyenne a été révélée inférieure à la dose efficace rapportée pour le [18F]FDG. En conclusion, ces résultats précliniques et cliniques ont révélé une grande spécificité de ce radiopharmaceutique pour les tissus lymphoprolifératifs. De plus, cela pourrait être un outil robuste pour quantifier plus précisément la maladie, même avec des processus inflammatoires, évitant ainsi les résultats faussement positifs, et une approche innovante pour l'imagerie des maladies lymphoprolifératives caractérisées par une faible activité mitotique. / Although PET using [18F]FDG has proved to be useful for diagnosis and therapy monitoring in patients with lymphoma, the specificity of [18F]FDG uptake has been critically questioned because of its dependence on glucose metabolism, which may indiscriminately increase in benign conditions such as inflammatory or infectious processes. Considering these drawbacks, an adenine nucleoside analogue was developed as a novel PET imaging probe ([18F]fludarabine). An efficient and fully automated radiosynthesis has been implemented and, subsequently preclinical studies in xenograft murine models (follicular cell lines: RL7 and DOHH-2, multiple myeloma (MM): RPMI8226, central nervous system (CNS) lymphoma: MC116) were conducted with this novel 18F-radiopharmaceutical in parallel with [18F]FDG. The pattern of the radioactivity distribution in selected organs confirmed the tumor-specific targeting. In a follicular lymphoma model, we evaluated its robustness during rituximab therapy and demonstrated - the treatment did not interfere with its uptake - a stronger correlation between quantitative values extracted from this 18F-radiopharmaceutical and histology when compared to [18F]FDG-PET. Accordingly, this PET tool may be considered as a promising approach for detecting the persistence of residual disease during or after rituximab-like treatment. Furthermore, its behaviour in turpentine-induced inflammatory process showed significantly weaker uptake in inflammation when compared to [18F]FDG. In MM, the role of [18F]FDG-PET remains limited because of its lack of sensitivity for detecting diffuse bone marrow involvement, small skull lesions due to the physiological [18F]FDG uptake in brain. Our data suggested that [18F]fludarabine-PET might represent an alternative and perhaps more specific modality for MM imaging. In our latest study, on xenograft brain tumors, this novel PET probe revealed significantly divergent responses between CNS lymphoma and glioblastoma (GBM), while [18F]FDG demonstrated overlap between the groups. A first in man study, was undertaken, for an initial diagnosis, where 10 untreated patients were enrolled with either B-cell chronic lymphocytic leukemia (CLL) or diffuse large B-cell lymphoma (DLBCL). Successive partial body scans were acquired for 250 min after i.v. injection with an activity of 4 MBq/kg. The results with conventional modalities (CT and/or [18F]FDG-PET) have also been investigated. The study was also designed to estimate its radiation dose for major organs. In DLBCL patients, increased uptake was observed in sites considered abnormal by CT and [18F]FDG; in two patients discrepancies were observed in comparison with [18F]FDG. In CLL patients, the uptake coincided with sites expected to be involved and displayed a significant uptake in hematopoietic bone marrow. No uptake was observed, whatever the disease group, in the cardiac muscle and brain. Moreover, its mean effective dose was below the effective dose reported for [18F]FDG. In conclusion, these preclinical and clinical findings revealed a great specificity of this 18F-radiopharmaeutical for lymphoma tissues. Furthermore, it might well be a robust tool for correctly quantifying the disease, even with inflammatory processes, thus avoiding the false-positive results, and an innovative approach for imaging lymphoid neoplasms with low mitotic activity.