Τα μυελοδυσπλαστικά σύνδρομα (ΜΔΣ) είναι μια ετερογενής ομάδα επίκτητων κλωνικών διαταραχών του πολυδύναμου αρχέγονου αιμοποιητικού κυττάρου, που χαρακτηρίζονται από κυτταροπενίες στο περιφερικό αίμα, μορφολογικές και λειτουργικές διαταραχές των αιμοποιητικών κυττάρων και αυξημένο κίνδυνο εκτροπής σε οξεία μυελογενή λευχαιμία (ΟΜΛ). Η επίπτωση των ΜΔΣ ποικίλλει σημαντικά μεταξύ διαφόρων χωρών με βάση δεδομένα από διάφορες μελέτες. Δεν υπήρχαν δημοσιευμένα επιδημιολογικά δεδομένα σχετικά με την επίπτωση των ΜΔΣ στον ελλαδικό χώρο μέχρι την πραγματοποίηση αυτής της διατριβής. Ασθενείς και μέθοδοι: Στο πρώτο σκέλος της διατριβής καταγράφηκαν όλοι οι ασθενείς που διαγνώστηκαν με ΜΔΣ κατά την 20-ετή περίοδο 1/1/1990-31/12/2009 στην περιοχή της Δυτικής Ελλάδας. Τα δεδομένα συλλέχθηκαν από τους ιατρικούς φακέλους των ασθενών με τεκμηριωμένη διάγνωση ΜΔΣ από ειδικό αιματολόγο ή αιμοπαθολογοανατόμο, και από τα τέσσερα νοσοκομεία την περιοχή της Δυτικής Ελλάδας (Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών, Γενικό Νοσοκομείο «Ο Άγιος Ανδρέας», Θεραπευτήριο «Ολύμπιον» και Γενικό Νοσοκομείο Μεσολογγίου). Καταγράφηκαν τα δημογραφικά και κλινικά χαρακτηριστικά των ασθενών με ΜΔΣ και δημιουργήθηκε μια ηλεκτρονική βάση δεδομένων, επί της οποίας έγινε περιγραφική στατιστική ανάλυση. Καταγράφηκε επίσης η ημερομηνία αρχικής διάγνωσης και η ημερομηνία θανάτου ή τελευταίας επαφής/εξέτασης και έγινε ανάλυση επιβίωσης. Εκτιμήθηκε η παρουσία συννοσηρών παθήσεων κατά τη διάγνωση και υπολογίστηκαν οι δείκτες συννοσηρότητας Charlson Comorbidity Index και MDS-CI. Η επίπτωση και ο επιπολασμός των ΜΔΣ υπολογίστηκαν για την περιοχή της Δυτικής Ελλάδας που απαρτίζεται από τους νομούς Αχαΐας, Ηλείας και ΜΔΣ. Το δεύτερο σκέλος της διατριβής είναι μια μελέτη ασθενών-μαρτύρων που πραγματοποιήθηκε στο Πανεπιστημιακό Νοσοκομείο Πατρών και στο Γενικό Νοσοκομείο «Ο Άγιος Ανδρέας». Ελήφθησαν συνεντεύξεις βάσει ερωτηματολογίου από ασθενείς με ΜΔΣ και ομάδα μαρτύρων με αναλογική εξομοίωση ως προς φύλο και ηλικία. Η ομάδα των μαρτύρων συγκροτήθηκε από ασθενείς που υπεβλήθησαν σε επέμβαση καταρράκτη στην Οφθαλμολογική Κλινική του Πανεπιστημίου Πατρών. Η συνέντευξη βασίστηκε σε ερωτηματολόγιο αναφορικά με την οικογενειακή κατάσταση, την περιοχή κατοικίας, το επάγγελμα, το οικογενειακό ιστορικό καρκίνου, την επαγγελματική έκθεση σε χημικά, το κάπνισμα, την κατανάλωση αλκοόλ, τη διατροφή, την έκθεση σε οικιακούς παράγοντες κινδύνου, τις δραστηριότητες ελεύθερου χρόνου, την ακτινοβολία για διαγνωστικούς σκοπούς και τυχόν προηγηθέν ψυχοπιεστικό γεγονός. Και τα δύο ερευνητικά σκέλη της διατριβής έλαβαν την έγκριση του Επιστημονικού Συμβουλίου των συμμετεχόντων νοσοκομείων. Η στατιστική ανάλυση έγινε με το στατιστικό πρόγραμμα IBM SPSS Statistics (έκδοση 20.0). Αποτελέσματα: Συνολικά καταγράφηκαν 855 ασθενείς με ΜΔΣ. Η ανθεκτική αναιμία (RA) ήταν η πιο κοινή υποκατηγορία και στα δύο φύλα με βάση την ταξινόμηση τόσο κατά FAB όσο και κατά WHO. Οι κατηγορίες Del(5q) και RARS ήταν πιο συχνές στις γυναίκες, ενώ η CMML-D στους άνδρες. Η τρισωμία 8 ήταν η πιο κοινή μονήρης κυτταρογενετική ανωμαλία. Η αδρή μέση ετήσια επίπτωση ΜΔΣ ήταν 6 ανά 100.000 κατοίκους ηλικίας ≥15 ετών (όλες οι κατηγορίες ΜΔΣ κατά FAB), ενώ ήταν 4,8 ανά 100.000 όταν εξαιρέθηκαν CMML και RAEB-T. Η αδρή επίπτωση ήταν υψηλότερη στις αγροτικές από ό,τι στις αστικές περιοχές, αλλά αυτό το εύρημα δεν επιβεβαιώθηκε μετά από προτύπωση κατά ηλικία. Η προτυπωμένη κατά ηλικία μέση ετήσια επίπτωση ΜΔΣ ήταν 7,9 ανά 100.000 στους άνδρες και 3,4 ανά 100.000 στις γυναίκες. Παρατηρήθηκε μια συνεχώς αυξανόμενη επίπτωση ΜΔΣ, που ουσιαστικά αντιπροσωπεύει μια αύξηση στις περιπτώσεις RA και RARS κατά τη διάρκεια της μελετηθείσας περιόδου. Η μέση επιβίωση των ασθενών με ΜΔΣ ήταν 39,8 μήνες και η διάμεση επιβίωση ήταν 22,4 μήνες. Η επιβίωση συσχετίστηκε σημαντικά με την ηλικία κατά τη διάγνωση και με την προγνωστική κατηγορία κατά IPSS. Η μονοπαραγοντική ανάλυση με βάση το μοντέλο του Cox έδειξε ότι η ηλικία, η κατηγορία κατά FAB, η βαρύτητα της αναιμίας, της λευκοπενίας, της ουδετεροπενίας και της θρομβοπενίας και τα επίπεδα φερριτίνης και LDH στον ορό συσχετίστηκαν με τη συνολική επιβίωση. Η παρουσία καρδιακής ανεπάρκειας ή/και νεφρικής νόσου κατά τη διάγνωση φάνηκε να επηρεάζει σημαντικά την επιβίωση, ενώ οι δείκτες συννοσηρότητας Charlson Comorbidity Index και MDS-CI δε φάνηκε να συσχετίζονται με την επιβίωση των ασθενών με ΜΔΣ στην παρούσα μελέτη. Διακόσιοι είκοσι τέσσερις ασθενείς συμμετείχαν στη μελέτη ασθενών-μαρτύρων (126 ασθενείς με ΜΔΣ και 98 μάρτυρες). Οι ασθενείς και οι μάρτυρες εξομοιώθηκαν αναλογικά ως προς το φύλο και την ηλικία. Το οικογενειακό ιστορικό αιματολογικής νεοπλασίας ή/και συμπαγούς όγκου συσχετίστηκαν σημαντικά με τα ΜΔΣ. Η επαγγελματική έκθεση σε φυτοφάρμακα, ιδιαίτερα εντομοκτόνα και ζιζανιοκτόνα, συσχετίστηκε σημαντικά με τον κίνδυνο εμφάνισης ΜΔΣ. Η κατανάλωση κρέατος ≥5 φορές την εβδομάδα, αυγών ≥3 φορές την εβδομάδα και ποσότητας αλκοόλ ≥15 ποτά (ισοδύναμα) την εβδομάδα επίσης συσχετίστηκαν με ΜΔΣ. Ωστόσο, οι μοναδικοί παράγοντες που διατήρησαν τη στατιστική σημαντικότητά τους στην πολυπαραγοντική ανάλυση ήταν το οικογενειακό ιστορικό κακοήθειας (συμπαγούς όγκου) (p=0.014) και η έκθεση σε φυτοφάρμακα (p<0.0001). / “Epidemiologic features, temporal trends of epidemiological indices and investigation of risk factors for myelodysplastic syndromes in Western Greece” Background: “Myelodysplastic syndromes (MDS) are a heterogenous group of acquired clonal disorders of the bone marrow, characterized by cytopenias, morphologic and functional abnormalities of hematopoietic cells, and a high risk of transformation to acute myeloid leukaemia (AML)”. The incidence of MDS varies significantly among countries according to different studies. Published epidemiologic data for MDS in Greece were not available by the time this study was conducted. Objective: The objective of the present study was to describe the demographic and clinical features of the patients diagnosed with MDS in Western Greece during the period 1990-2009, to estimate the incidence of MDS and its temporal trends throughout this period, as well as to investigate risk factors for MDS in the same area. This was achieved in two main parts: the first part involved the creation of a local MDS registry and a subsequent descriptive study, and the second part was a case-control study. Patients and methods: In the first part of the thesis, all patients diagnosed with MDS in Western Greece during the 20-year-period 1/1/1990-31/12/2009 were registered. Data were retrieved from the medical records of patients with a documented diagnosis of MDS, performed by an expert hematologist and/or hematopathologist, in all four hospitals situated in the geographical area of Western Greece. Demographic and clinical features of patients with MDS were collected and an electronic database was created, upon which descriptive analysis was performed. Date of diagnosis and date of death or date of last contact were also registered and survival analysis was performed. Comorbidities at diagnosis were also evaluated and comorbidity indices (Charlson Comorbidity Index and MDS-CI) were calculated. Incidence and prevalence of MDS was calculated for the well-defined geographical area of Western Greece, which consists of the prefectures Achaia, Ilia and Etolia-Akarnania. Temporal trend of incidence rates was also studied. The second part of the thesis was a hospital-based case-control study conducted in two hospitals in the city of Patras, Greece. MDS prevalent cases and proportionally age- and gender-matched controls were interviewed. The group of controls consisted of patients who were operated for cataract at the Department of Ophthalmology. The interview was based on a questionnaire regarding marital status, area of residence, profession, family history of cancer, occupational exposure to chemicals, smoking, alcohol consumption, nutrition, exposure to domestic risk factors, leisure activities, radiation for diagnostic purposes and stressful life events. Both parts of the study were approved by the Ethical and Scientific Committee of the participating hospitals. Statistical analysis was performed with the statistical software IBM SPSS Statistics 20.0. Results: A total of 855 patients with newly diagnosed MDS were identified. Refractory anemia was the most common subtype in both FAB and WHO classification systems and in both genders. Del(5q) and RARS were more commonly encountered among females and CMML-D among males. Trisomy 8 was the most common single cytogenetic abnormality. The crude mean annual incidence rate of MDS was 6.0 per 100,000 inhabitants aged ≥15 years old (all subtypes according to FAB), and it was 4.8 per 100,000 when CMML and RAEB-T were excluded. Crude incidence rate was higher in rural than in urban areas, but this finding was not confirmed after age-standardization. Age-standardized mean annual incidence rate of MDS was 7.9/100,000 in men and 3.4/100,000 in women. A continuously increasing incidence rate of MDS was observed, which essentially represented an increase in cases of RA and RARS throughout the study period. Mean survival of patients with MDS was 39.8 months and median survival was 22.4 months. Survival was significantly associated with age at diagnosis and with IPSS prognostic category. Univariate analysis with Cox regression model revealed that age, FAB subtype, the degree of anemia, leucopenia, neutropenia, thrombocytopenia, serum ferritin and LDH levels were all associated with overall survival. The presence of heart failure and/or chronic kidney disease at diagnosis proved to significantly affect survival, whereas Charlson Comorbidity Index and MDS-CI were not shown to correlate with survival. Two hundred and twenty four patients participated in the case control study (126 MDS cases and 98 controls). Cases and controls were proportionally matched by age and gender. Family history of hematologic malignancy and family history of solid tumour were significantly associated with MDS. Occupational exposure to agricultural chemicals, and especially herbicides and insecticides, was significantly associated with MDS. Consumption of meat ≥5 times a week and eggs ≥3 times a week, and alcohol consumption ≥15 drinks (alcohol equivalents) a week were also associated with MDS. In multivariate analysis, the only factors which eventually retained their statistical significance were family history of solid malignancy (p=0.014) and exposure to agricultural chemicals (p<0.0001).

Η κυτταρομετρία ροής (Flow Cytometry–FC), είναι μία σύγχρονη αυτοματοποιημένη τεχνική ανάλυσης των φυσικοχημικών χαρακτηριστικών των κυττάρων και των σωματιδίων, η οποία επιτρέπει την μεμονωμένη μέτρησή τους, καθώς διέρχονται σε νηματική ροή από ένα σταθερό σημείο, που προσπίπτει ακτίνα laser. Η ουσιαστική χρήση της FC είναι η προσφορά της σε διάγνωση και παρακολούθηση ασθενών με νοσήματα που συνοδεύονται από παρουσία παθολογικών κυττάρων σε διάφορα βιολογικά υγρά ή και στερεούς ιστούς κατάλληλα επεξεργασμένους. Το αποτέλεσμα της κυτταρομετρικής ανάλυσης είναι μία πληθώρα μετρήσεων φθορισμού, καθώς και των δύο μετρήσεων πρόσθιου (Forward Scatter–FS) και πλάγιου (Side Scatter-SS) σκεδασμού, που εξαρτώνται από τα φυσικά χαρακτηριστικά κάθε κυττάρου. Μετά την ανάλυση των δεδομένων από τον Ηλεκτρονικό Υπολογιστή (Η/Υ) του κυτταρομετρητή, τα αποτελέσματα παρουσιάζονται υπό τη μορφή μονοπαραμετρικών ή πολυπαραμετρικών κατανομών. Στην ανάλυση που χρησιμοποιήθηκε (με χρήση 5 φθοριοχρωμάτων), ο κυτταρομετρητής ροής παρήγαγε 7 τιμές για κάθε ένα από τα 30.000 κύτταρα περίπου που μετρήθηκαν σε κάθε πρωτόκολλο. Με τη χρήση των Η/Υ μπορούμε να αναλύσουμε γρήγορα και αξιόπιστα όλον αυτό τον μεγάλο όγκο δεδομένων εφαρμόζοντας μοντέλα βάσεων δεδομένων. Η βασική δομή του σχεσιακού μοντέλου δεδομένων αναπαριστάται με ένα πίνακα, στον οποίο αποθηκεύονται δεδομένα, σε στήλες και γραμμές, τα οποία αφορούν μία συγκεκριμένη οντότητα. Οι σχέσεις των πινάκων περιγράφουν τoν τρόπο σύνδεσης διαφορετικών οντοτήτων, οι οποίες συνδυαστικά δημιουργούν λογικούς πίνακες, που με τη σειρά τους περιγράφουν πιο σύνθετες οντότητες. Κατά αυτόν τον τρόπο μπορούμε να κάνουμε περαιτέρω συγκρίσεις μεταξύ των εξετάσεων των ασθενών, που ίσως καταλήξουν σε ευνοϊκά συμπεράσματα, όσον αφορά την πρόγνωση και την θεραπεία κυρίως των νεοπλασματικών νοσημάτων του αίματος. Ο ρόλος της FC σε αιματολογικά νοσήματα όπως τα μυελοδυσπλαστικά σύνδρομα (ΜΔΣ) είναι ακόμα υπό διερεύνηση. Τα ΜΔΣ είναι νοσήματα με σημαντική κλινική και αιματολογική ετερογένεια, κάτι που καθιστά σαφή την ανάγκη μαζικής ανάλυσης των δεδομένων τους, για την αναγνώριση ομοιόμορφων υποομάδων με κοινά γνωρίσματα και άρα ενός πληροφοριακού μοντέλου ανάλυσης που θα διευκολύνει την λήψη των κατάλληλων θεραπευτικών επιλογών.Η παρούσα εργασία ασχολείται με τις πολυπαραμετρικές εξετάσεις των ΜΔΣ, την πληροφορία των οποίων είναι ικανή να παρέχει η FC. Θα γίνει προσπάθεια να καταγραφούν αναλυτικά όλα τα απαραίτητα βήματα, έτσι ώστε σε δεύτερο χρόνο να αναλυθεί μαζικά όλη αυτή η πληροφορία μέσω ενός σχεσιακού μοντέλου βάσεων δεδομένων. / Flow cytometry (Flow Cytometry-FC), is a modern automated technical analysis of the physicochemical characteristics of cells and particles, which allows the individual measuring them as they pass in threaded flow from a fixed point, incident beam laser. The effective use of FC is offering a diagnosis and monitoring of patients with diseases associated with the presence of abnormal cells in various biological fluids and solid tissues or processed properly. The results of cytometric analysis is a plethora of fluorescence measurements and measurements of both anterior (Forward Scatter-FS) and lateral (Side Scatter-SS) dispersion, which depends on the physical characteristics of each cell. After analyzing the data from the PC (H / H) on the cytometer, the results presented in the form monoparametric or multi parameter distributions. The analysis used (using fluorochrome 5), the flow cytometer produced 7 values ​​for each of the 30,000 or so which cells were measured in each protocol. By using the H / H can be analyzed quickly and reliably throughout this large volume of data by applying models of databases. The basic structure of relational data model is represented by a table that stores data in columns and rows, which relate to a specific entity. Relations ton of tables describing how to connect different entities, which in combination create logical tables, which in turn describe more complex entities. In this way we can make further comparisons between the examinations of patients, which may lead to favorable conclusions regarding the prognosis and treatment of neoplastic diseases, especially blood. The role of FC in hematological diseases such as myelodysplastic syndromes (MDS) is still under investigation. MDS is a disease with significant clinical and haematological heterogeneity, which makes clear the need for mass analysis of their data, to identify subgroups with common standard features and thus an informative analysis model that will facilitate the adoption of appropriate therapeutic epilogon.I present work dealing with multivariate MDS tests, information which is capable of providing the FC. I try to record in detail all the necessary steps so that a second time to analyze all this mass of information via a relational database model.

Em nosso trabalho anterior mostramos que dentre as citocinas analisadas, os níveis do mRNA de LIF nas células estromais pediátricas, de SMD e de SMD-LMA foram maiores quando comparados às células estromais de crianças saudáveis. No presente estudo, observamos um aumento tempo dependente nos níveis da proteína LIF após adição de SFB em todas as células analisadas (células estromais de crianças saudáveis, de SMD e de SMD-LMA). O envolvimento de p38, ERK e JNK na expressão LIF nestas células foi determinado pelo uso de inibidores dos membros das proteínas quinase ativadas por mitógenos: ERK (PD98059), p38 (SB302580) e JNK (SP600125) os quais inibiram a produção de LIF nas células estromais de crianças saudáveis, após estas serem estimuladas por SFB. No entanto, os níveis da expressão de LIF-induzido por soro nas células estromais de SMD e de SMD-LMA tratadas com SB302580 (p38) foram significativamente diminuídos, em comparação com a inibição observada no tratamento com PD98059 e SP600125 (p <0001, teste ANOVA). Em adição analisamos as formas fosforiladas de p38 e ERK, após 48hs na ausência ou na presença de soro por diferentes tempos. Níveis de atividade de ERK e do p38 foram inicialmente elevados na ausência de soro. A atividade de p38 foi sustentada após tratamento com SFB, entretanto, ERK apresentou uma variação de atividade durante o tratamento. Sugerimos que a sinalização das MAPKs (p38, ERK e JNK), em resposta a fatores de crescimento presentes no soro, parece desempenhar um papel importante na expressão da LIF em células estromais de crianças saudáveis, mas a sinalização do p38 parece ser funcionalmente mais importante nas mielodisplasias ou naquelas associadas à LMA / Our previous report showed that among the cytokines analysed, LIF mRNA levels in stromal cells from pediatric MDS and MDS-AML were higher as compared to those found in healthy stromal cells. In the present study, we have observed an increased protein LIF levels in a time dependent manner after FCS stimulation in all stromal cells analysed (MDS, MDS-AML and healthy children) and the involvement of p38, ERK and JNK pathways in the LIF expression in these cells was determined. In stromal cells from two healthy children, LIF production was equally inhibited in a dose dependent manner after FCS stimulation by mitogen-activated protein kinase (MAPKs) members inhibitors: ERK (PD98059), p38 (SB302580) and JNK (SP600125). However, in MDS and MDS-AML stromal cells, the levels of LIF-induced by serum, were significantly decreased by SB302580, as compared with the inhibition observed by treatment with PD98059 and SP600125 (p <0,001, ANOVA test). In addition we have analysed the presence of p38 and ERK phosphorylated forms in stromal cells, after 48hs of serum starvation or in the presence of FCS for different times. Activated ERK and p38MAPK levels were initially elevated in the absence of serum. p38MAPK activation was sustained after treatment with FCS, whereas ERK presented a variation of the activated forms during treatment. We suggest that the signalling of the MAPKs (p38, ERK and JNK) in response to growth factors present in the serum, seems to play an important role in the LIF expression by stromal cells of healthy children, but p38 MAPK signalling appears to be functionally more important in MDS and MDS-AML

Accurate localization of breakpoints and deleted regions on chromosome 7 in bone marrow cells of patients is an essential step in identifying genes involved in tumor transformation of a cell. In case of hematological malignancies usually oncogenes and tumor suppressor genes are activated or deleted by a change in the arrangement of genetic material. Aberration of chromosome 7, total or partial loss of chromosome, especially long arms 7q, are among the recurrent cytogenetic abnormalities in patients with myeloid diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Aberrations of chromosome 7 are an important prognostic marker occurring in 8-10% de novo MDS and AML and in 40-50% treated MDS / AML. For a detailed analysis of chromosome 7 breakpoints and aberrations, samples of 51 adult patients diagnosed with AML / MDS were examined using conventional and molecular cytogenomic methods. In our testing group we demonstrated a separate 7q deletion in one patient (2%) and an isolated monosomy of chromosome 7 in six patients (12%). Aberration of chromosome 7 detected in combination with another change was found in 17 cases (33%) and 27 patients (53%) had complex karyotype changes including chromosome 7. The most frequent breakpoint was 7q22. In 26 patients we proved a deletion...

Myelodysplastic syndrome (MDS) is a clonal disease of hematopoesis resulting from damage to hematopoietic stem cells. The most common chromosomal aberration in patients with MDS is deletion of the long arms of chromosome 5, del(5q). The aim of this study is to analyse unbalanced aberrations of chromosome 5 in MDS patients, to compare the extent of 5q deletion in groups of patients with isolated del(5q) and with del(5q) in complex karyotypes, and to study the effect of the extent of del(5q) on overall survival and prognosis of the disease. We combined cytogenomic methods to examine 88 bone marrow samples from patients with MDS and del(5q) confirmed by conventional banding methods. Del(5q) was present in the karyotype as an isolated aberration in 31 patients (35,2 %), in combination with one other clonal aberration in 9 patients (10,2 %), and as part of complex karyotypes in 48 patients (54,6 %). Patients with complex karyotypes had a lower overall survival than patients with isolated del(5q). The occurrence of complex karyotypes was associated with a large extent of 5q deletion. When both the occurrence of complex karyotypes and the extent of 5q deletion were considered, only karyotype complexity had a significant effect on patients' overall survival. The extent of the deletion does not affect...

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.

[ES] Los síndromes mielodisplásicos (SMD) constituyen un grupo heterogéneo de enfermedades de naturaleza clonal caracterizadas por presentar una hematopoyesis ineficaz, citopenias y riesgo variable de evolución a leucemia mieloide aguda (LMA) secundaria. En la última década, las nuevas tecnologías de secuenciación masiva han revelado que más del 80 % de pacientes con SMD presenta mutaciones somáticas y que éstas pueden agruparse en diversas categorías en función de las rutas biológicas que se vean alteradas. Además, se ha visto que existen patrones de concurrencia y exclusión entre estas categorías de mutaciones. La adquisición secuencial y la concurrencia entre estas mutaciones desencadenan, en parte, el desarrollo de la enfermedad y genera la heterogeneidad clínica característica de los SMD. Las mutaciones en factores de splicing aparecen a menudo simultáneamente con mutaciones en reguladores epigenéticos como es el caso de los genes U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) y Ten-eleven translocation 2 (TET2) que se encuentran co-mutados en un 13 % de los casos. A pesar de su prevalencia, los efectos de la concurrencia en las mutaciones en U2AF1 y TET2 no han sido estudiados. Por ello, en esta tesis nos propusimos estudiar esta cooperación cruzando, en primer lugar, dos líneas mutantes de ratón generadas mediante el sistema de edición genética CRISPR/Cas9. El efecto de estas alteraciones sobre la hematopoyesis de las tres líneas mutantes, U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/-, fue examinado mediante el hemograma, citometría de flujo (CF), análisis morfológicos, ensayos de Unidades Formadoras de Colonias (CFU) y estudios funcionales como el trasplante hematopoyético. Para finalizar, se realizó un análisis transcriptómico mediante secuenciación de ARN (ARN-seq) para detectar los posibles cambios en el patrón de splicing entre las líneas mutantes y los controles. La línea mutante U2af1mut/+ no presentó ninguna alteración destacable de la hematopoyesis ni en ratones jóvenes (12-13 semanas) ni envejecidos (2 años). Sin embargo, sus células madre y progenitoras hematopoyéticas (HSPC) fueron incapaces de injertar en la médula ósea de ratones trasplantados. En el caso de los ratones mutantes Tet2-/-, observamos un incremento de células mieloides, esplenomegalia, aumento del compartimento LSK (HSPC con inmunofenotipo Linaje- Sca-1+ c-kit+) y, en los experimentos de trasplante, una capacidad de reconstitución hematopoyética superior a la de los controles. Por último, la cooperación de ambas alteraciones en la línea doble mutante U2af1mut/+ Tet2-/-, no mostró un efecto sinérgico entre ellas. Así pues, se detectaron variaciones en los progenitores mieloeritroides y un aumento significativo de células mieloides y LSK. No obstante, igual que ocurría con la línea U2af1mut/+, las HSPC no producían prendimiento en los ratones trasplantados. A pesar de las alteraciones observadas, ninguna de las tres líneas mutantes desarrollaba SMD ni fallecía antes que los controles. Respecto al análisis transcriptómico, el salto de exón fue el evento de splicing alternativo observado con mayor frecuencia en las líneas U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/-. Únicamente un 6.6 % del total de genes que presentaba eventos de splicing alternativo fueron coincidentes en las tres líneas mutantes. A pesar de que en el análisis bioinformático se detectaron alteraciones en las rutas biológicas relacionadas con el ciclo celular, en los ratones U2af1mut/+, y el daño al ADN, en las líneas U2af1mut/+ y U2af1mut/+ Tet2-/-, en la validación mediante CF no se encontraron variaciones respecto a los controles. Para concluir, nuestros datos sugieren que, a pesar de producirse alteraciones en la hematopoyesis, la cooperación entre la mutación en U2af1 y la pérdida de Tet2 es insuficiente para iniciar SMD en ratón. / [CA] Les síndromes mielodisplàstiques (SMD) constituïxen un grup heterogeni de malalties de naturalesa clonal caracteritzades per presentar una hematopoesi ineficaç, citopènies i risc variable d'evolució a leucèmia mieloide aguda (LMA) secundària. En l'última dècada, les noves tecnologies de seqüenciació massiva han revelat que més del 80 % de pacients amb SMD presenta mutacions somàtiques i que aquestes poden agrupar-se en diverses categories en funció de les rutes biològiques que es vegen alterades. A més, s'ha vist que hi ha patrons de concurrència i exclusió entre aquestes categories de mutacions. L'adquisició seqüencial i la concurrència entre aquestes mutacions desencadenen, en part, el desenvolupament de la malaltia i genera l'heterogeneïtat clínica característica de les SMD. Les mutacions en factors de splicing apareixen sovint simultàniament amb mutacions en reguladors epigenètics com és el cas dels gens U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) i Ten-eleven translocation 2 (TET2) que es troben co-mutats en un 13 % dels casos. A pesar de la seua prevalença, els efectes de la concurrència en les mutacions en U2AF1 i TET2 no han sigut estudiats. Per això, en aquesta tesi ens vam proposar estudiar aquesta cooperació creuant, en primer lloc, dos línies mutants de ratolí generades per mitjà del sistema d'edició genètica CRISPR/Cas9. L'efecte d'aquestes alteracions sobre l'hematopoesi de les tres línies mutants, U2af1mut/+, Tet2-/- i U2af1mut/+ Tet2-/-, va ser examinat per mitjà de l'hemograma, citometría de flux (CF), anàlisis morfològiques, assajos d'Unitats Formadores de Colònies (CFU) i estudis funcionals com el trasplantament hematopoètic. Per últim, es va realitzar l'anàlisi transcriptòmic per mitjà de seqüenciació d'ARN (ARN-seq) per a detectar els possibles canvis en el patró de splicing entre les línies mutants i els controls. La línia mutant U2af1mut/+ no va presentar cap alteració destacable de l'hematopoesi ni en ratolins jóvens (12-13 setmanes) ni envellits (2 anys). No obstant això, les seues cèl·lules mare i progenitores hematopoetiques (HSPC) van ser incapaços d'empeltar en la medul·la òssia de ratolins trasplantats. En el cas dels ratolins mutants Tet2-/-, observarem un increment de cèl·lules mieloides, esplenomegàlia, augment del compartiment LSK (cèl·lules mare amb inmunofenotip Llinatge- Sca-1+ c-kit+) i, en els experiments de trasplantament, una capacitat de reconstitució hematopoética superior a la dels controls. Finalment, la cooperació d'ambdues alteracions en la línia doble mutant U2af1mut/+ Tet2-/-, no va mostrar un efecte sinèrgic entre elles. Així, doncs, es van detectar variacions en els progenitors mieloeritroids i un augment significatiu de cèl·lules mieloides i LSK. No obstant això, igual que ocorria amb la línia U2af1mut/+, les HSPC no produïen empelt en els ratolins trasplantats. A pesar de les alteracions observades, cap de les tres línies mutants desenvolupava SMD ni moria abans que els controls. Respecte a l'anàlisi transcriptòmic, el salt d'exó va ser l'esdeveniment de splicing alternatiu observat amb major freqüència en les línies U2af1mut/+, Tet2-/- i U2af1mut/+ Tet2-/-. Únicament un 6.6 % del total de gens que presentava esdeveniments de splicing alternatiu van ser coincidents en les tres línies mutants. Encara que en l'anàlisi bioinformàtica es van detectar alteracions en les rutes biològiques relacionades amb el cicle cel·lular, en els ratolins U2af1mut/+, i el dany a l'ADN, en les línies U2af1mut/+ i U2af1mut/+ Tet2-/-, en la validació per mitjà de CF no es van trobar variacions respecte als controls. Per a concloure, les nostres dades suggerixen que, a pesar de produir-se alteracions en l'hematopoesi, la cooperació entre la mutació en U2af1 i la pèrdua de Tet2 és insuficient per a iniciar SMD en ratolí. / [EN] Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal malignancies characterized by ineffective hematopoiesis, cytopenia and a variable risk of progression to secondary acute myeloid leukemia (AML). In the last decade, next-generation sequencing technologies have deciphered that more than 80 % of MDS patients have somatic mutations and that those can be grouped into several categories depending on which biological routes have been altered. Furthermore, it has been observed that there are concurrency and exclusion patterns among these mutation categories. The sequential acquisition and the concurrency between these driver mutations trigger, in part, the development of the disease and generate the clinical heterogeneity characteristic of MDS. The splicing factor mutations often occur simultaneously with mutations in epigenetic regulators such as the U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) and Ten-eleven translocation 2 (TET2) genes, which are found co-mutated in 13 % of cases. Despite their prevalence, the effects of concurrence in mutations in U2AF1 and TET2 have not been studied. Consequently, in this thesis we aim to study this cooperation. Firstly, we crossed two mutant mouse lines that were previously generated using the CRISPR/Cas9 gene editing system. The effects of these alterations on hematopoiesis in the three mutant lines, U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/-, was examinated by the blood counts, flow cytometry (FC), morphological analysis, Colony Forming Units assays (CFU) and functional studies such as the hematopoietic transplantation. Finally, transcriptomic analysis was peformed by RNA sequencing (RNA-seq) to detect possible splicing pattern changes between mutant lines and control samples. U2af1mut/+ mutant line did not present any remarkable alteration of hematopoiesis in either in young (12-13 weeks) or aged (2 years) mice. However, their hematopoietic stem and progenitor cells (HSPC) were unable to engraft into the bone marrow of transplanted mice. In the case of Tet2-/- mutant mice, we observed an increase of myeloid cells, splenomegaly, an increased LSK compartment (HSPC: Lineage- Sca-1+ c-kit+) and an enhanced ability, relative to wild-type, to reconstitute hematopoiesis in transplantation assays. Finally, the cooperation of both alterations in U2af1mut/+ Tet2-/- double mutant line did not show a synergistic effect between them. Nonetheless, the myeloerythroid progenitors were altered and also myeloid and LSK cells were increased. However, as in the U2af1mut/+ line, HSPC did not produce any engraftment in transplanted mice. Despite the observed alterations, none of the three mutant lines developed MDS or die earlier than control mice. Regarding the transcriptomic analysis, exon skipping was the most frequently observed alternative splicing event in the U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/- lines. Only 6.6 % of the total number of genes showing alternative splicing events were coincident in the three mutant lines. Although the bioinformatic analysis revealed alterations in biological pathways related to the cell cycle in the U2af1mut/+ mice and DNA damage in the U2af1mut/+ and U2af1mut/+ Tet2-/- lines, the validation by CF found no variations with respect to the controls. In conclusion, our data suggest that, despite alterations in hematopoiesis, the cooperation between U2af1 mutation and Tet2 loss is insufficient to initiate MDS in mice. / Martínez Valiente, C. (2022). Desarrollo y caracterización de un modelo de ratón doble mutante en U2af1 y Tet2 para el estudio de los Síndromes Mielodisplásicos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/187749 / TESIS

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.

As síndromes mielodisplásicas (SMD) se caracterizam por terem uma hematopoese displásica, citopenias e pelo risco de progressão para leucemia mielóide aguda. O diagnóstico baseia-se na clínica e nos achados citomorfológicos da medula óssea (MO) e citogenéticos. Na fase inicial ou quando a MO é hipocelular o diagnóstico é difícil e a citogenética frequentemente é normal. A imunofenotipagem (IMF) tem sido cada vez mais utilizada nos casos de SMD em adultos e pouco explorada na SMD pediátrica. Os nossos objetivos foram: estudar os casos de SMD e doenças correlatas (LMA relacionada à SMD: LMA-rMD; leucemia mielomonocítica crônica: LMMC e leucemia mielomonocítica juvenil: LMMJ) em adultos e crianças, associando os dados clínicos e laboratoriais aos obtidos pela IMF, que utilizou um painel de anticorpos monoclonais para as várias linhagens medulares. No período compreendido entre 2000 e 2010 foram estudados 87 pacientes (64 adultos e 23crianças) oriundos do HUPE/UERJ e IPPMG/UFRJ e 46 controles (23 adultos e 20 crianças). Todos os doentes realizaram mielograma, biópsia óssea, citogenética, citoquímica e estudo imunofenotípico. Segundo os critérios da OMS 50 adultos foram classificados como SMD, 11 como LMA-rMD e 3 LMMC. Entre as crianças 18 eram SMD, 2 LMA e 3 LMMJ. Os pacientes adultos com SMD foram divididos em alto risco (n = 9; AREB-1 e AREB-2) e baixo risco (n=41; CRDU, CRDM, CRDM-SA, SMD-N e SMD-5q-). As crianças com SMD em CR (n=16) e AREB (n = 2). Anormalidades clonais recorrentes foram encontradas em 22 pacientes adultos e em 7 crianças. Na análise da IMF foi utilizada a metodologia da curva ROC para a determinação dos valores de ponto de corte a fim de identificar os resultados anormais dos anticorpos monoclonais nos pacientes e nos controles, permitindo determinar a sensibilidade e especificidade desses em cada linhagem. A IMF foi adequada para a análise em todos os pacientes e 3 ou mais anormalidades foram encontradas. A associação da IMF aumentou a sensibilidade da análise morfológica na linhagem eritróide de 70 para 97% nos adultos e de 59 para 86% nas crianças; na linhagem granulocítica de 53 para 98% nos adultos e de 50 para 100% nas crianças. Nos monócitos, onde a morfologia não foi informativa, mostrou uma sensibilidade de 86% nos adultos e 91% nas crianças. Enquanto que na linhagem megacariocítica, não analisada pela IMF, a morfologia mostrou uma sensibilidade de 95% nos adultos e 91% nas crianças. Na população de blastos foi expressiva a ausência de precursores linfóide B (em 92% dos adultos e em 61% das crianças). Os resultados observados nas crianças com SMD foram semelhantes aos encontrados nos adultos. Em conclusão, nossos resultados mostraram que a IMF é um método complementar ao diagnóstico da SMD e doenças correlatas tanto em adultos quanto em crianças podendo contribuir para o reconhecimento rápido e precoce dessas enfermidades, devendo ser incorporado aos procedimentos de rotina diagnóstica. / As síndromes mielodisplásicas (SMD) se caracterizam por terem uma hematopoese displásica, citopenias e pelo risco de progressão para leucemia mielóide aguda. O diagnóstico baseia-se na clínica e nos achados citomorfológicos da medula óssea (MO) e citogenéticos. Na fase inicial ou quando a MO é hipocelular o diagnóstico é difícil e a citogenética frequentemente é normal. A imunofenotipagem (IMF) tem sido cada vez mais utilizada nos casos de SMD em adultos e pouco explorada na SMD pediátrica. Os nossos objetivos foram: estudar os casos de SMD e doenças correlatas (LMA relacionada à SMD: LMA-rMD; leucemia mielomonocítica crônica: LMMC e leucemia mielomonocítica juvenil: LMMJ) em adultos e crianças, associando os dados clínicos e laboratoriais aos obtidos pela IMF, que utilizou um painel de anticorpos monoclonais para as várias linhagens medulares. No período compreendido entre 2000 e 2010 foram estudados 87 pacientes (64 adultos e 23crianças) oriundos do HUPE/UERJ e IPPMG/UFRJ e 46 controles (23 adultos e 20 crianças). Todos os doentes realizaram mielograma, biópsia óssea, citogenética, citoquímica e estudo imunofenotípico. Segundo os critérios da OMS 50 adultos foram classificados como SMD, 11 como LMA-rMD e 3 LMMC. Entre as crianças 18 eram SMD, 2 LMA e 3 LMMJ. Os pacientes adultos com SMD foram divididos em alto risco (n = 9; AREB-1 e AREB-2) e baixo risco (n=41; CRDU, CRDM, CRDM-SA, SMD-N e SMD-5q-). As crianças com SMD em CR (n=16) e AREB (n = 2). Anormalidades clonais recorrentes foram encontradas em 22 pacientes adultos e em 7 crianças. Na análise da IMF foi utilizada a metodologia da curva ROC para a determinação dos valores de ponto de corte a fim de identificar os resultados anormais dos anticorpos monoclonais nos pacientes e nos controles, permitindo determinar a sensibilidade e especificidade desses em cada linhagem. A IMF foi adequada para a análise em todos os pacientes e 3 ou mais anormalidades foram encontradas. A associação da IMF aumentou a sensibilidade da análise morfológica na linhagem eritróide de 70 para 97% nos adultos e de 59 para 86% nas crianças; na linhagem granulocítica de 53 para 98% nos adultos e de 50 para 100% nas crianças. Nos monócitos, onde a morfologia não foi informativa, mostrou uma sensibilidade de 86% nos adultos e 91% nas crianças. Enquanto que na linhagem megacariocítica, não analisada pela IMF, a morfologia mostrou uma sensibilidade de 95% nos adultos e 91% nas crianças. Na população de blastos foi expressiva a ausência de precursores linfóide B (em 92% dos adultos e em 61% das crianças). Os resultados observados nas crianças com SMD foram semelhantes aos encontrados nos adultos. Em conclusão, nossos resultados mostraram que a IMF é um método complementar ao diagnóstico da SMD e doenças correlatas tanto em adultos quanto em crianças podendo contribuir para o reconhecimento rápido e precoce dessas enfermidades, devendo ser incorporado aos procedimentos de rotina diagnóstica.