As síndromes mielodisplásicas (SMD) constituem um grupo heterogêneo de doenças hematológicas de origem clonal, caracterizado por hematopoese ineficaz, citopenia e risco de evolução para leucemia mieloide aguda (LMA). As anormalidades citogenéticas adquiridas são marcadores prognósticos bem estabelecidos em SMD. No entanto, a técnica de citogenética metafásica apresenta limitações, incluindo baixa resolução e necessidade de divisão celular, sendo que defeitos cromossômicos podem não ser detectados. Tecnologias baseadas em microarranjo (array) de DNA, como o Single Nucleotide Polymorphism Array (SNP-A), são importantes para avaliação do genoma normal e neoplásico. O SNP-A foi desenvolvido para o estudo de todo o genoma, apresenta uma resolução superior a citogenética metafásica convencional, pode ser realizado em células na interfase, e detecta alterações cromossômicas não visualizadas pela citogenética metafásica. Além disso, o SNPA fornece dados de genotipagem para detecção de perda neutra de heterozigose, também denominada de dissomia uniparental somática. Regiões cromossômicas com deleção, perda neutra de heterozigose ou ganho são comuns em pacientes com neoplasias hematológicas e sugeriu genes candidatos a supressores de tumor e oncogenes. O objetivo do presente estudo foi a caracterização da coorte de pacientes com suspeita clínica de SMD e o uso integrado do método de citogenética convencional e SNP-A no serviço de hematologia da nossa instituição na investigação de alterações citogenéticas em pacientes com SMD e doenças relacionadas. Durante o período do estudo, foram recebidas um total de 114 amostras de pacientes com suspeita clínica de SMD. A análise clínica, morfológica e citogenética permitiu confirmar o diagnóstico de SMD ou doenças relacionadas em 43 pacientes (SMD [n=34], SMD/NMP [n=5], LMA com alterações mielodisplásicas [n=4]). Vinte e um pacientes foram classificados como citopenia idiopática de significado indeterminado (CISI) e 50 indivíduos apresentaram outros diagnósticos. SNP-A foi realizado em 17 pacientes com SMD e doenças relacionadas. Dentre os pacientes selecionados para o SNP-A, anormalidades cromossômicas foram observadas em 6/17 (35%) casos pelo cariótipo convencional e em 8/17 (47%) casos pela técnica de SNP-A. SNP-A não detectou quatro alterações cromossômicas previamente identificadas pela citogenética convencional: duas translocações balanceadas e duas alterações numéricas. SNP-A confirmou os demais achados identificados pela citogenética convencional e detectou um total de 32 novas lesões (1 ganho, 19 perdas e 12 UPDs) em 6 pacientes com SMD ou doenças relacionadas. SNP-A pode complementar a citogenética convencional na detecção de anormalidades cromossômicas em neoplasias mieloides. / Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic diseases, characterized by inefficient hematopoiesis, peripheral blood cytopenias and a risk to progress to acute myeloid leukemia (AML). Acquired chromosomal abnormalities have prognostic value in MDS. However, metaphase cytogenetics has some limitations including low resolution and the requirement of cell division, and chromosomal abnormalities may not be detected. New technologies based on array, the Single Nucleotide Polymorphism Array (SNP-A), are able to evaluate the whole genome. The SNP-A has superior resolution compared to metaphase cytogenetics, may be used in interphase cells, and may detect chromosomal abnormalities not detected by metaphase cytogenetics. In addition, the SNP-A read-out includes genotyping calls and hybridization signal strength, corresponding to gene copy number, allowing detecting copy neutral loss of heterozigosity (CN-LOH), also known as uniparental dissomy (UPD). Deletions, copy neutral loss of heterozigosity or gain are frequent in patients with haematopoietic neoplasms and has already suggested the location of tumor suppressor genes and oncogenes. The aim of this study was to characterize the cohort of patients with clinical suspicion of MDS and to establish the integrative use of the conventional cytogenetic and the SNP-A in the investigation of chromosomal abnormalities in patients with MDS and related diseases followed at our institution. The clinical, morphological and cytogenetic evaluation allowed us to confirm the diagnosis of MDS or related disease in 43 patients (MDS [n=34], MDS/MPN [n=5], AML with myelodysplastic changes [n=4]). Twenty-one patients were diagnosed with idiopathic cytopenia with undetermined significance (ICUS) and 50 patients had other diagnosis. SNP-A were performed in 17 patients with MDS and related disease. Chromosomal abnormalities were observed in 6/17 (35%) cases by metaphase cytogenetics, and in 8/17 (47%) of the cases by SNP-A. SNP-A did not detected two balanced translocations and two numerical alterations previously observed by metaphase cytogenetics. SNP-A confirmed all the other findings observed by metaphase cytogenetics and SNP-A detected a total of 32 new lesions (1 gain, 19 losses and 12 UPDs) in 6 MDS and related diseases. SNP-A may complement metaphase cytogenetics to improve the detection of chromosomal abnormalities in myeloid neoplasms.

Les syndromes myélodysplasiques (MDS) sont un groupe de pathologies myéloïdes caractérisées par une hématopoïèse inefficace. Le rôle du microenvironnement médullaire (MM) dans l’histoire naturelle de ces pathologies reste incertain. Des anomalies du MM ont été décrites au cours des myélodysplasies et des modèles murins récemment publiés font penser qu’une altération du MM pourrait jouer un rôle dans le déclenchement et/ou l’évolution de ces maladies.Nous avons tenté de développer un modèle in vivo récapitulant l’histoire naturelle des myélodysplasies par des xénogreffes chez des souris NSG et NSG-S. Le faible taux de prise de greffe nous a amenés à développer un modèle in vitro de co-culture en 2D. Ce modèle est une bonne alternative pour les études de nouvelles stratégies thérapeutiques pour les patients atteints de myélodysplasies.Au cours de ce travail, nous avons également réalisé une étude systématique du stroma médullaire de patients atteints de syndromes myélodysplasiques dans le but d’identifier les anomalies fonctionnelles et moléculaires des cellules souches mésenchymateuses (CSMs), cellules centrales du MM pour leur interaction avec les cellules souches hématopoïétiques (CSHs).Les CSMs de MDS ont une clonogénécité diminuée. Nous n’avons pas observé de modification significative de leurs capacités de différenciation en ostéoblastes, adipocytes et chondrocytes ni dans leur capacité à supporter une hématopoïèse normale. Les CSMs de MDS présentent des modifications au niveau épigénétique et transcriptionnel pouvant expliquer l’altération des relations observées grâce à de l’imagerie enregistrée entre les CSMs de MDS et les CSHs dans un modèle de co-culture en 3D.Ces résultats montrent que les CSMs de MDS ont des modifications fonctionnelles et moléculaires et que ces anomalies perturbent leur relation avec les CSHs. / Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid pathologies characterized by an impaired hematopoiesis. The role of the bone marrow microenvironment (BMM) remains unclear in the natural history of these diseases. Abnormalities of the BMM have been observed in myelodysplasia and a recent published murine model implies that alterations of the BMM could play a role in the trigger/progression of these diseases.Firstly, we tried to develop an in vivo model of MDS in NSG and NSG-S mice. The low rate of engraftment pushed us to develop a 2D co-culture model in vitro. This model is a good alternative to test new therapeutic strategies for MDS patients.In this study, we analysed mesenchymal stromal cells (MSCs) from the bone marrow of pretreated MDS patients in order to identify the functional and molecular abnormalities in those cells of the BMM, central for their interactions with the hematopoietic stem cells (HSCs).MDS MSCs have an impaired clonogenic capacity. We didn’t observed modifications of their differentiation toward osteogenic, adipogenic and chondrogenic pathways and capacity to support of a normal hematopoiesis. MDS MSCs display epigenetic and transcriptomic modifications that could explain the alteration of the relationships between these cells and HSCs observed in imagery in a 3D co-culture model.These results showed that MDS MSCs have functional and molecular abnormalities and that these alterations could impair their relationship with HSCs.

Cancer remains one of the most feared diseases affecting the modern world. Second to heart disease, it is the largest cause of deaths, affecting one in three persons. Cancer cells are formed when normal, healthy cells become damage, losing their normal regulatory mechanism that control cell growth. There are many different types and progression of these cancer cells that determine the type of treatment a patient receives. The primary focus of this dissertation is to propose three studies of anticancer agents. In Chapter one, a CpG-lytic peptide conjugate was designed to target receptors on the cell membrane to concentrate the lytic peptide around the cells to cause triggered cell death, in the treatment of Myelodysplastic Syndromes (MDS). This conjugate act like monoclonal antibodies in that the molecular size is too large to enter the cell, therefore it targets the TLR9 receptors expressed extracellularly in precancer cells in MDS. Chapter two, focuses on the screening of anticancer agents used in targeted therapy. It provides a general scheme applied to the synthesis of a combinatorial library of primary amines used as small-molecule drugs coupled unto a solid support bead (Positional Scanning Library Method) to screen for biological effects on various types of cancers. Chapter three address the issue of radiotherapy treatments, one of the most widely used treatment of cancer. To improve the efficacy of conventional radiation therapy and reduce the cytotoxicity of healthy tissue, High-Dose Rate brachytherapy (HDR) may be used as a stand-alone treatment or after surgery to prevent the recurrence of cancer cells. To design and provide studies of these brachytherapy beads, a model was developed by coupling a chelating agent DOTA onto the surface of macrobeads that coordinated to Europium (III) in efforts to mimic the radiolabeling with a radioactive metal. These brachytherapy beads will be used to conduct in vitro studies in the treatment of local cancers with massive concentrations of radiation without damaging surrounding healthy tissue.

名古屋大学博士学位論文 学位の種類:博士(医療技術学) (課程) 学位授与年月日:平成20年9月30日

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression. The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered. Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS. Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action. Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders. We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites. These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

Η αιμοποιητική φωλαιά (hematopoietic stem cell niche) περιέχει οστεοβλάστες, οι οποίοι ρυθμίζουν τη φυσιολογική αιμοποίηση. Ωστόσο, λίγα στοιχεία είναι γνωστά, έως τώρα, για το ρόλο των οστεοβλαστών στη διαδικασία της αιμοποίησης σε ασθενείς με Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ). Το ΜΔΣ, αποτελεί μια ετερογενή ομάδα κλωνικών αιματολογικών διαταραχών, με αυξημένο κίνδυνο εκτροπής προς Οξεία Μυελογενή Λευχαιμία (ΟΜΛ). Μελέτες σε ex-vivo συστήματα καλλιεργειών (co-cultures) περιγράφουν την επίδραση των μεσεγχυματικών κυττάρων (“feeder cells”) στο δυναμικό πολλαπλασιασμού, στη μεταναστευτική ικανότητα, καθώς και στη διατήρηση (stemness) των αρχέγονων αιμοποιητικών κυττάρων (HSCs) φυσιολογικών δοτών. Η μελέτη αυτή στοχεύει στη διερεύνηση των βιολογικών χαρακτηριστικών των οστεοβλαστών από ασθενείς με ΜΔΣ καθώς και τις αλληλεπιδράσεις φυσιολογικών HSCs και οστεοβλαστών ασθενών με ΜΔΣ. Για το σκοπό αυτό δημιουργήθηκε ένα σύστημα δισδιάστατης καλλιέργειας (2-D culture system) χρησιμοποιώντας οστεοβλάστες που παρήχθησαν από μεσεγχυματικά κύτταρα μυελού των οστών (human marrow mesenchymal stem cells-MSCs). Τα MSCs απομονώθηκαν από το μυελό των οστών ασθενών με ΜΔΣ και υγιών δοτών και καλλιεργήθηκαν σε κατάλληλο θρεπτικό μέσο. Ακολούθησε επαγωγή της διαφοροποίησης των MSCs, μετά από συνεχόμενες καλλιέργειες σε οστεοβλάστες. Στη μελέτη χρησιμοποιήθηκαν 13 δείγματα μυελού των οστών από ασθενείς με ΜΔΣ (6 RA, 3 RAEBI, 2 RAEBII, 1 5q- και 1 υποπλαστικό MDS) και 8 δείγματα μυελού φυσιολογικών μαρτύρων όμοιας ηλικίας. Για τη μελέτη της επίδρασης των οστεοβλαστών από ασθενείς με ΜΔΣ στην αιμοποίηση χρησιμοποιήθηκαν φυσιολογικά HSCs από κινητοποιημένο περιφερικό αίμα υγιών δοτών (mPB, n=4), τα οποία τοποθετήθηκαν πάνω στους ήδη εγκατεστημένους οστεοβλάστες (osteoblast confluent monolayer cultures). Τα MSCs και οι οστεοβλάστες που αναπτύχθηκαν ελέγχθηκαν μορφολογικά και ανοσοφαινοτυπικά, με τη χρήση μικροσκοπίας και κυτταρομετρίας ροής αντίστοιχα. Μονοπύρηνα κύτταρα από δείγματα κινητοποιημένου περιφερικού αίματος υγιών δοτών τοποθετήθηκαν στο δισδιάστατο καλλιεργητικό σύστημα, σε καλλιεργητικό υλικό αιμοποιητικών κυττάρων, χωρίς την εξωγενή προσθήκη κυτταροκινών. Με τη χρήση κυτταρομετρίας ροής ελέγχθηκε η έκφραση των μορίων που σχετίζονται με την προσκόλληση των αιμοποιητικών κυττάρων στην αιμοποιητική φωλαιά καθώς και την εγκατάσταση και διατήρησή τους σε αυτή. Ο έλεγχος έγινε στις 36 ώρες και τις 7 ημέρες συγκαλλιέργειας και αφορούσε τα μόρια CXCR4, το οποίο ρυθμίζει την άμεση πρόσδεση των HSCs στην φωλαιά κατά τη διαδικασία του “homing”, CD49d (Very Late Antigen-4- VLA4) και CD49e (Very Late Antigen-5- VLA5), τα οποία παρέχουν σήματα επιβίωσης ή προάγουν την ενεργοποίηση μιας φάσης ηρεμίας (quiescence) στα HSCs μετά την είσοδο τους στη φωλαιά (localization). Η έκφραση των μορίων αυτών μελετήθηκε στους υποπληθυσμούς των CD34+, CD34+/CD38+ και CD34+/CD38- κυττάρων. Παράλληλα εκτιμήθηκε το ποσοστό (συχνότητα) των CD34+ αιμοποιητικών κυττάρων καθώς επίσης και η προσκόλλησή τους στους οστεοβλάστες. Μετά τη συγκαλλιέργεια, οι οστεοβλάστες που προήλθαν από υγιείς δότες προκάλεσαν τον πολλαπλασιασμό των CD34+ κυττάρων των φυσιολογικών αιμοποιητικών κυττάρων που τοποθετήθηκαν πάνω στο εγκατεστημένο στρώμα των οστεοβλαστών (3-fold και 9-fold αύξηση στις 36ώρες και τις 7ημ., αντίστοιχα). Αύξηση επίσης, παρατηρήθηκε (2 fold αύξηση) στα CD34+ κύτταρα στις συγκαλλιέργειες των 36h, φυσιολογικών HSCs με οστεοβλάστες που παρήχθησαν από ασθενείς με ΜΔΣ, ενώ καμία διαφορά δεν παρατηρήθηκε μεταξύ των διαφορετικών υποτύπων ΜΔΣ. Στις 7 ημέρες συγκαλλιέργειας από την άλλη, δεν 12 παρατηρήθηκε καμία διαφορά στη συχνότητα εμφάνισης ενός πιο άωρου φαινοτύπου των φυσιολογικών HSCs που αναπτύχθηκαν σε οστεοβλάστες από ασθενείς με χαμηλού κινδύνου ΜΔΣ (low risk MDS). Αντιθέτως, τα CD34+ κύτταρα αυξήθηκαν κατά πολύ (16- fold αύξηση), όταν φυσιολογικά HSCs, τοποθετήθηκαν σε οστεοβλάστες ασθενών με υψηλού κινδύνου ΜΔΣ (high risk MDS). Επιπλέον, παρατηρήθηκε αύξηση της έκφρασης των μορίων CXCR4, CD49d και CD49e στα CD34+ κύτταρα μετά από συγκαλλιέργεια φυσιολογικών HSCs και οστεοβλαστών από υγιείς δότες, συγκριτικά με τα επίπεδα έκφρασης των μορίων αυτών πριν την τοποθέτηση τους στο σύστημα συγκαλλιέργειας. Η αύξηση της έκφρασης του μορίου CXCR4 ήταν λιγότερο εμφανής στην περίπτωση συγκαλλιέργειας των φυσιολογικών HSCs με οστεοβλάστες από ασθενείς με ΜΔΣ, όπου η μεγαλύτερη διαφορά παρατηρήθηκε στο σύστημα που περιείχε τους οστεοβλάστες από ασθενείς χαμηλού κινδύνου ΜΔΣ (3- και 1,7- fold αύξηση στις 7ημέρες καλλιέργειας με οστεοβλάστες από υγιείς δότες και χαμηλού κινδύνου ΜΔΣ ασθενείς, αντίστοιχα). Το πρότυπο έκφρασης των μορίων CD49d και CD49e ήταν όμοιο στα κύτταρα που τοποθετήθηκαν τόσο σε οστεοβλάστες προερχόμενους από υγιείς δότες, όσο και οστεοβλάστες από ΜΔΣ ασθενείς. Ο φαινότυπος, τόσο όσον αφορά τα μορφολογικά όσο και τα ανοσοφαινοτυπικά χαρακτηριστικά, των MSCs ήταν ίδιος και στις δυο ομάδες μελέτης, ενώ η διαφοροποίηση των MSCs προς οστεοβλάστες ήταν όμοια τόσο στα MSCs που προήλθαν από φυσιολογικούς δότες όσο και σε αυτά που προήλθαν από ασθενείς με ΜΔΣ, δείχνοντας παρόμοια έκφραση των ειδικών οστεοβλαστικών πρωτεϊνών αλλά και της διαδικασίας της ενασβεστοποίησης. Σύμφωνα με τα αποτελέσματα που λάβαμε, οι οστεοβλάστες από υγιείς δότες προώθησαν την αύξηση του ποσοστού των προγονικών αιμοποιητικών κυττάρων και οδήγησαν στην επαγωγή της έκφρασης του μορίου CXCR4, ενός πολύ σημαντικού μορίου για τη μετανάστευση, την εγκατάσταση αλλά και την ανάπτυξη. Ωστόσο, η διαφορετική δραστηριότητα, τόσο όσον αφορά το ποσοστό των CD34+ όσο και την έκφραση του μορίου CXCR4, όταν τα φυσιολογικά αιμοποιητικά κύτταρα συγκαλλιεργήθαν με οστεοβλάστες που προήλθαν από ασθενείς με ΜΔΣ, οδηγεί στην υπόθεση ότι υπάρχει μεταβολή στη λειτουργία των οστεοβλαστών, οπότε προβλέπεται και μια επακόλουθη αλλαγή στη ρύθμιση της εγκατάστασης των HSC στην αιμοποιητική φωλαιά, στους ασθενείς με ΜΔΣ. / The hematopoietic stem cell niche contains osteoblasts that regulate normal hematopoiesis. However, little is known about the role of osteoblasts in MDS hematopoiesis so far. Myelodysplastic syndrome comprises a heterogeneous group of clonal stem cell disorders with dismal prognosis and difficulty in their therapeutic approach, which is characterized by ineffective hematopoiesis. It appears with dysplastic hematopoietic cells, peripheral blood cytopenias and high risk of evolution to acute myeloid leukemia (AML). Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-cell contact has a significant impact on the expansion, migratory potential and “stemness” of hematopoietic stem cells. In this study, we investigated the biological characteristics of osteoblasts from MDS patients and the interactions between these cells and normal hematopoietic stem cells (HSCs). Osteoblasts were differentiated from marrow MSCs from 13 MDS patients (6 RA, 3 RAEBI, 2 RAEBII, 1 5q- and 1 hypoplastic MDS) and 8 age-matched healthy individuals. To study the effect of MDS osteoblasts on hematopoiesis, normal HSCs from mobilized peripheral blood from healthy individuals (n=4) were seeded onto osteoblast confluent monolayer cultures using a culture medium appropriate for the culture of HSCs, without the exogenous addition of cytokines. We studied the morphology and immunophenotype of MSCs and osteoblasts by microscopy and flow cytometric analysis, respectively. Cytometric analyses of homing associated molecules were performed 36h and 7d later. These molecules are CXCR4, which regulates the direct adhesion of HSCs to the bone marrow niche during “homing”, CD49d (Very Late Antigen-4- VLA4) and CD49e (Very Late Antigen-5- VLA5), which produce survival signals or promote the maintenance of a quiescent state for HSCs after entering the stem cell niche (localization). We investigated the expression of these molecules in CD34+, CD34+/CD38+ and CD34+/CD38- cell populations. Furthermore we studied the frequency of CD34+ hematopoietic cells and also their ability to adhere osteoblasts.Osteoblasts from healthy individuals increased the frequency of CD34+ cells by 3- and 9-fold increase in normal hematopoietic cells after 36h and 7d co-cultures respectively. A 2-fold increase was also seen in CD34+ cells when normal HSCs grown on MDS-osteoblasts for 36h and no difference was seen between the MDS subtypes. When the culture period was extended to 7d, there was no change in the frequency of immature phenotype of normal HSCs in osteoblast cultures from low-risk MDS patients. In contrast, CD34+ cells increased several fold (16-fold increase) when normal HSCs were cultured on high-risk MDS 14 osteoblasts, twice the values obtained in osteoblast co-cultures from healthy individuals and low risk patients. The expression of adhesion molecules CXCR4, CD49d and CD49e on CD34+ cells from normal HSCs was increased in co-cultures with osteoblasts from healthy individuals compared to the values obtained before culture (3-fold increase at 7d). The increase in CXCR4 expression was less pronounced in the presence of osteoblasts from MDS patients with the largest difference being found in low-risk MDS patients (1,7-fold increase at 7d). The expression pattern of CD49d and CD49e was identical between cells grown on MDS- and normal- osteoblast co-cultures.The morphological and immunophenotypical analysis of MSCs show the same results for the two study groups, while the differentiation of MSCs to osteoblasts was similar for both healthy individuals and MDS patients, after having similar expression of bone specific proteins and mineralization activity. According to our data, osteoblasts from healthy individuals promoted the expansion of immature hematopoietic progenitors and induced the cell surface expression of CXCR4, an important molecule in HSCs homing, retention and development. However, the different expression of CXCR4 and the change in frequency of CD34+ cells that were detected when normal HSCs co-operated with MDS-osteoblasts, suggests alteration in osteoblast function and the subsequent regulation of the HSC residency in the niche in MDS patients compared with healthy individuals.

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-17Bitstream added on 2014-06-13T20:54:04Z : No. of bitstreams: 1 freitas_pc_me_sjrp.pdf: 528091 bytes, checksum: d630cd8e1a7a4fdc34c7e9200a36b8b5 (MD5) / Síndromes Mielodisplásicas (SMD) compreendem um conjunto heterogêneo de doenças hematopoéticas caracterizadas por hematopoese ineficaz, que geralmente apresentam citopenias no sangue periférico, medula óssea hipercelular, diferenciação celular displásica e propensão ao desenvolvimento de leucemia mielóide aguda. São classificadas em oito tipos e a incidência anual é estimada entre dois e 12 casos por 100.000 pessoas da população em geral e em até 50 casos por 100.000 indivíduos com idades superiores a 60 anos. A análise cromossômica das células da medula óssea dos doentes ao diagnóstico detecta alterações diretamente relacionadas com o prognóstico em aproximadamente 50% dos casos. Alguns genes também foram relacionados à etiologia e prognóstico das mielodisplasias. O gene FOXO3, um supressor de tumor, embora não estudado anteriormente em SMD, é um dos genes que mais se expressam no tecido hematopoético normal. Alterações neste gene poderiam resultar em hematopoese anormal, pois já foram relacionadas a outros tipos de câncer, com mutações descritas no éxon 1. O objetivo deste trabalho foi estudar células da medula óssea de doentes com SMD de qualquer tipo, ao diagnóstico, para investigar a presença de alterações cromossômicas e de mutações no éxon 1 do FOXO3. A análise citogenética foi realizada em metáfases submetidas ao bandamento GTG, obtidas de culturas de curta duração de células da medula, sem estimulação mitogênica. Para a análise molecular foi extraído o DNA, realizada a amplificação gênica pela Reação em Cadeia da Polimerase e realizado o sequenciamento direto do éxon 1. Entre os 25 casos analisados, três (12%) apresentaram alterações cromossômicas clonais isoladas: deleção intersticial do braço longo do cromossomo 5; monossomia do cromossomo 21 e monossomia do cromossomo 22. Todas puderam ser relacionadas... / Myelodysplastic syndrome (MDS) constitute a heterogeneous group of hematopoietic diseases characterized by ineffective hematopoiesis usually with peripheral blood cytopenia, hypercellular bone marrow, dysplastic differentiation and a tendency to evolve to acute myeloid leukemia. They are classified in eight categories by the World Health Organization. The annual incidence is estimated at between two and 12 cases per 100,000 individuals in the general population and up to 50 cases per 100,000 of over 60-year olds. A chromosomal analysis of bone marrow cells at diagnosis identifies changes directly related to prognosis in approximately 50% of cases. Additionally, some genes are also associated to the etiology and prognosis of myelodysplasia. Although not previously studied in respect to MDS, a tumor suppressor, FOXO3, is one of the most commonly expressed genes in normal hematopoietic tissue. Changes in this gene could therefore result in abnormal hematopoiesis, as mutations described in exon 1 have already been associated with other types of cancer. The aim of this study was to investigate chromosomal alterations and mutations in exon 1 of FOXO3 in bone marrow cells from patients diagnosed with any type of MDS. Cytogenetic analysis was performed on metaphases submitted to GTG banding, obtained from short-term cultures of bone marrow cells without mitogenic stimulation. To evaluate mutations in the FOXO3 gene, DNA was extracted from the bone marrow, gene amplification was achieved by polymerase chain reaction and direct sequencing was performed. Of the 25 cases analyzed, three (12%) showed clonal chromosomal abnormalities in isolation characterized as the interstitial deletion of the long arm of chromosome 5, monosomy 21 and monosomy 22. All were correlated to the diagnosis and/or prognosis of patients. No mutations were detected in exon 1, but the 159C>T polymorphism was detected... (Complete abstract click electronic access below)

Orientador: Irene Gyöngyver Heidemarie Lorand-Metze / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T10:34:04Z (GMT). No. of bitstreams: 1 Vido_JoyceRico_M.pdf: 733870 bytes, checksum: d5a56d9b376e9b0ddadbf12bf5c2b264 (MD5) Previous issue date: 2011 / Resumo: A contagem de blastos na medula óssea (MO) é um parâmetro essencial para a classificação e prognóstico das síndromes mielodisplásicas (SMD). No entanto, neste grupo de doenças clonais há um alto grau de atipias de células hematopoiéticas da MO podendo ser difícil classificar com precisão os blastos mielóides. Nosso objetivo foi investigar se a análise de imagem computadorizada de esfregaços de citologia de rotina corados com May-Grünwald-Giemsa seria capaz de caracterizar estas células. Precursores mielóides imaturos foram digitalizados e as imagens segmentadas de forma interativa usando esfregaços de MO: 30 casos de SMD recém-diagnosticados (15 RCMD, 11 AREB, 4 AR / ARSA) e 19 casos de MO normal. A classificação morfológica das células foi feita por consenso de dois observadores. A distribuição da cromatina nuclear foi analisada por variaveis de morfometria geomatrica, variáveis derivadas da matriz de co-ocorrência e dimensão fractal (DF) para verificar sua utilidade na classificação destas células. Entre as 15 variáveis estudadas, todas, exceto área nuclear e homogeneidade local foram capazes de distinguir os blastos de promielócitos normais. Precursores atípicos mielóides que morfologicamente lembravam mieloblastos apresentaram valores intermediários entre blastos e promielócitos de acordo com 5 variáveis e foram classificados como promielócitos por mais 7 variáveis. A área nuclear não foi significativamente diferente entre os diversos tipos de células. Precursores mielóides atípicos imaturos da MO podem ser difíceis de classificar corretamente em citologia de rotina. Eles podem ser blastos com maturação anormal ou promielócitos atípicos. Como mudanças de textura da cromatina nuclear refletem a remodelação dinâmica da cromatina, a análise das variáveis obtidas pode ser útil para classificar objetivamente precursores imaturos mielóides nas SMD / Abstract: Bone marrow (BM) blast count is an essential parameter for classification and prognosis of myelodysplastic syndromes (MDS). However, in this group of clonal disorders a high degree of atypias in bone marrow hemopoietic cells may be found so that it may be difficult to quantify precisely myeloid blasts. Our aim was to investigate whether computerized image analysis of routine cytology would be able to characterize these cells. In May-Grünwald-Giemsa stained BM smears of 30 newly diagnosed MDS (15 RCMD, 11 RAEB, and 4 RA/RARS) patients and 19 cases of normal BM, blasts, promyelocytes and atypic myeloid precursors were digitalized and interactively segmented. The morphologic classification of the cells was done by consensus of two observers. Nuclear morphometry and texture features derived from the co-occurrence matrix and fractal dimension (FD) were calculated. Among the 13 variables studied, all except nuclear area and local homogeneity were able to distinguish blasts from promyelocytes. Atypic myeloid precursors that morphologically resembled myeloblasts showed intermediate values between blasts and promyelocytes according to 5 variables and were classified as promyelocytes by another 7 variables. Nuclear area was not significantly different among the cells. BM atypical immature myeloid precursors may be difficult to classify correctly in routine cytology. They could be abnormally maturing blasts or atypical promyelocytes. As nuclear texture changes reflect chromatin remodeling dynamics, the analysis of the variables obtained by image analysis may be useful to objectively classify immature myeloid precursors in MDS / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientador: Irene Lorand-Metze / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T20:45:18Z (GMT). No. of bitstreams: 1 Reis-Alves_SuiellenCarvalho_M.pdf: 2640206 bytes, checksum: 0fe8b3a89ccee8a9cd543d9d74f965c3 (MD5) Previous issue date: 2009 / Resumo: A análise multiparamétrica por citometria de fluxo é útil para o diagnóstico diferencial de Síndromes Mielodisplásicas (SMD) nos casos com poucos elementos displásicos na medula óssea (MO) e cariótipo normal. Vários estudos têm relatado hipogranularidade dos granulócitos, anormalidades no padrão de expressão de antígenos, alterações nas quantidades e expressões anômalas nas células CD34+, assim como uma diminuição das células precursoras-B, além do aumento dos monócitos. No entanto, não há um consenso sobre qual o melhor painel a ser aplicado, pois, os painéis geralmente utilizados incluem um grande número de anticorpos monoclonais (AcMo). Neste estudo, a utilidade de um painel racionalizado (10 AcMo) foi avaliada, em combinações de quatro cores, na rotina laboratorial, permitindo o estudo de diversos parâmetros para o diagnóstico de SMD, podendo sugerir fatores prognósticos. Foi examinado MO de pacientes com diagnóstico recente de SMD. O diagnóstico foi baseado na contagem de sangue periférico, citologia de medula óssea e cariótipo. O critério OMS foi utilizado. Foram analisados: dispersão lateral da luz (side scatter -SSC) e padrão de maturação dos granulócitos e dos monócitos, além dos subtipos presentes na população de células CD34+, sendo estas características comparadas com a MO normal. As combinações de AcMo utilizadas foram: HLA-DR/CD14/CD45/CD33; CD16/CD34/CD45/CD13; CD19/CD34/CD45/CD117 e HLA-R/CD123/CD45/CD33. No mínimo 50.000 eventos/caso foram adquiridos. Este estudo incluiu 24 casos de MO normal e 54 casos de SMD com idade mediana de 62 anos (23-93). Os tipos OMS foram 2 casos de AR, 2 SMD del 5q, 25 CRDM, 8 CRDMSA, 7 AREB-I, 10 AREB-II. Os casos foram grupados em SMD com <5% de blastos (baixo risco) e SMD >5% de blastos (alto risco) para análise e comparados com MO normal. Foram detectadas 16 alterações: 4 nos precursores granulocíticos, 4 nos monócitos, 6 na população de blastos como o aumento das células CD34+, mieloblastos e células imaturas não definidas, diminuição dos precursores de células B, e 2 nas populações minoritárias. O total de número de alterações em casos com < 5% de blastos na MO foram de 6 (2-15) porcentagem de blastos na citologia (r= 0.38; p= 0.001), porcentagem de células CD34+ (r= 0.40; p< 0.001), células CD34+/CD13+ (r= 0.61; p< 0.0001), e com as células imaturas não linfóides CD34+/CD13- e CD34+/CD117- (r= 0.30 p=0.02 e r=0.55 p=0.0003, respectivamente). Os precursores de células-B (r=-0.39; p= 0.001) e a hemoglobina (r= -0.30; p= 0.001) diminuíram de acordo com a extensão do número de alterações. Houve correlação entre o número de alterações com o tipo OMS (r= 0.38; p=0.002) e o IPSS (r=0.27 p=0.02). Mesmo com o uso de um painel restrito de 10 AcMo, concluiu-se que este painel foi suficiente para fazer o diagnóstico em 91% dos casos, e permitiu detectar características associadas com a agressividade dos casos. / Abstract: Flow cytometric analysis is useful for the diagnosis of myelodysplastic syndromes (MDS) in cases of few dysplastic elements in bone marrow (BM) and a normal karyotype. Several studies have reported decreased side scatter (SSC) in the granulocytic gate, abnormalities in the maturation pattern antigens, number and abnormal co-expressions in CD34+ cells besides decreased of B-cell precursors and increased of monocytes. There is no uniformly accepted panel for these analyses that usually comprise a large number of monoclonal antibodies (MoAbs). We examined the utility of a small panel of MoAbs in four-color combinations, used routinely in our laboratory that allows the evaluation of several parameters for the diagnosis of MDS and may suggest prognostic factors. Bone marrow aspiration was performed in the diagnostic routine. The diagnosis was based on peripheral blood counts, BM morphology and karyotype. The WHO criteria were used. We examined: SSC and maturation pattern of granulocytes and monocytes; subsets present in the CD34+ population. These features were compared to the values found in normal BM. Combinations of MoAbs:HLA-DR/CD14/CD45/CD33; CD16/CD34/CD45/CD13; CD19/CD34/CD45/CD117; HLA-R/CD123/CD45/CD33. At least 50 000 events/case were acquired. Data were acquired in a FACS CaliburTM flow cytometer and the Paint-A-Gate software was used for data analysis. Normal BMs: 24 cases; MDS 54 cases. Median age: 62 years (23-93). WHO types: 2 RA, 2 MDS del 5q, 25 RCMD, 8 RCMD-RS, 7 RAEB-I, 10 RAEB-II. We could detect 16 alterations: 4 in granulocytic precursors, 4 in monocytes, increase in CD34+ cells, myeloblasts and not defined immature cells, decrease in B-cell precursors, changes in precursor lymphoid dendritic cells and basophilic precursors. The total number of changes in cases with <5% BM blasts was 6 (2-15) and in RAEB 8 (4-12). The number of alterations had a positive correlation with the WHO type, IPSS, %blasts (cytology) (r=0.38; p=0.001), %CD34+ cells (r=0.40; p=0.001), CD34+/CD13+ cells (r=0.61; p<0.0001), but also non-lymphoid immature cells (CD34+/CD13- r=0.30; p=0.02 e CD34+/CD117- r=0.55; p=0.0003). The B-precursors cells and hemoglobin decreased with the increase of number alterations (r=-0.39; p=0.001 and r=-0.30; p=0.01 respectively). We conclude that our panel, although small, was sufficient to make the diagnosis of MDS in 91% of our cases, and permitted to detect features associated with aggressive cases. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

As sÃndromes mielodisplÃsicas (SMD) representam um grupo heterogÃneo de doenÃas clonais que acometem a cÃlula precursora hematopoÃtica pluripotente, caracterizando-se por baixa contagem de cÃlulas no sangue perifÃrico, displasia em uma ou mais linhagens celulares, hematopoese ineficiente, alÃm do risco aumentado de progressÃo para leucemia mielÃide aguda. Embora a doenÃa possa acometer pacientes de outras faixas etÃrias, Ã mais frequente naqueles com idade avanÃada, com mÃdia ao diagnÃstico de 60 a 75 anos. As anormalidades cromossÃmicas sÃo observadas em aproximadamente 50% de todos os casos de SMD, estando, em alguns casos, relacionadas com achados clÃnicos e morfolÃgicos. O objetivo deste trabalho foi determinar, atravÃs da tÃcnica de FISH (hibridizaÃÃo in situ por fluorescÃncia), a frequÃncia de alteraÃÃes envolvendo os genes MLL, RB e TP53 em pacientes com SMD e associar estas alteraÃÃes com os achados citogenÃticos. Os casos inseridos no estudo foram oriundos do ambulatÃrio de SMD do Hospital UniversitÃrio Walter CantÃdio. Dos 33 pacientes selecionados, 17 pertenciam ao grupo com idade acima de 60 anos. 52% dos pacientes foram classificados, segundo a OMS, como citopenia refratÃria com displasia em mÃltiplas linhagens (CRDM) e 61% estratificados, segundo o IPSS, como de risco intermediÃrio 1 (INT-1). Um total de 78% dos pacientes apresentaram alteraÃÃes citogenÃticas, dentre eles 31% possuÃam cariÃtipos complexos (mais de 3 alteraÃÃes por metÃfase). A tÃcnica de FISH permitiu identificar em 18% dos pacientes alteraÃÃes envolvendo um dos trÃs genes avaliados. TrÃs pacientes apresentaram alteraÃÃo do gene TP53, sendo detectada em dois deles (registros 31 e 6) a deleÃÃo de um Ãnico alelo ou de ambos os alelos do gene, respectivamente, e no terceiro (registro 2), detectou-se a amplificaÃÃo do gene TP53, sendo estas alteraÃÃes nÃo visualizadas atravÃs da citogenÃtica clÃssica, por se tratar de um tÃcnica menos sensÃvel. Detectou-se em 6% dos pacientes (registros 7 e 22) rearranjo do gene MLL, no primeiro a FISH descartou a suposta deleÃÃo do gene alegada pela citogenÃtica, provando que o mesmo estava presente no genoma do paciente, porÃm de forma rearranjada e no segundo a citogenÃtica nÃo conseguiu demonstrar o rearranjo do gene. Quanto ao gene RB, a FISH permitiu identificar apenas um paciente (3%) com deleÃÃo de um dos alelos do gene, sendo esta alteraÃÃo tambÃm nÃo detectada pela citogenÃtica clÃssica. A FISH possibilitou identificar, durante a avaliaÃÃo do gene TP53, dois pacientes (registros 5 e 10) apresentando pelo menos 6 cÃpias extras do cromossomo 17, devendo essa alteraÃÃo se tratar de um pequeno clone hiperdiplÃide detectado parcialmente no primeiro paciente e nÃo detectado no segundo. Nos seis pacientes que apresentaram alteraÃÃo dos genes avaliados, a FISH proveu informaÃÃes que adicionaram, confirmaram ou alteraram o resultado previamente emitido pela citogenÃtica clÃssica, sendo estas uma das principais aplicaÃÃes desta tÃcnica devido sua alta sensibilidade quando comparada ao mÃtodo clÃssico. / Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders affecting the hematopoietic pluripotent cell, characterized by low cell counts in peripheral blood, dysplasia in one or more cell lines, inefficient hematopoiesis and increased risk of progression to acute myeloid leukemia. Although the disease can affect patients of other age groups, they are more frequent in those with advanced age with an average 60 to 75 years at diagnosis. Chromosomal abnormalities are observed in approximately 50% of all cases of MDS and are related with clinical and morphological findings. The aim of this study was to determine, through the technique of FISH (fluorescence in situ hybridization), the frequency of changes involving the MLL, RB, and TP53 genes in patients with MDS and associate these changes with cytogenetic findings. The cases included in the study were selected in the ambulatory of SMD from University Hospital Walter CantÃdio. Thirty three patients were selected, 17 had aged over 60 years. 52% of patients were classified, according to WHO criteria, as refractory cytopenia with dysplasia in multiple lineages (RCDM) and 61% stratified, according to IPSS, as intermediate risk 1 (INT-1). 78% of patients had abnormalities detected by cytogenetics, among them 31% had complex karyotypes (more than 3 changes per metaphase). 18% of patients had changes at least in one of the three genes valued in this study by FISH. Three patients showed alterations of TP53 gene, being detected in two patients (records 31 and 6) the deletion of a single allele or both alleles of the gene, respectively, and in the third (record 2), we detected amplification of TP53 gene, all this changes were not detected by classical cytogenetics, because it is a less sensitive technique. 6% of patients (records 7 and 22) had rearrangement of MLL gene. In the first case, FISH discarded the gene deletion alleged by cytogenetic, proving that it was present in the genome of the patient, but in a rearranged form, and in the second case cytogenetics failed to demonstrate rearrangement of the gene. For the RB gene, FISH identified only one patient (3%) with deletion of one allele of the gene, and this change was also not detected by classical cytogenetics. During evaluating the TP53 gene, FISH allowed identification of two patients (records 5 and 10) presenting at least six extra copies of chromosome 17, probably representing a small hyperdiploid clone partially detected in the first patient and not detected in the second . In the six patients who showed abnormalities of the genes analyzed, FISH has provided information that added, changed or confirmed the result previously given by classical cytogenetics, which are a major application of this technique due to its high sensitivity compared to the traditional method.