Στοχευμένος προσδιορισμός αλληλουχίας, με τεχνολογία δεύτερης γενιάς (Next Generation Sequencing), σε ασθενείς με μυελικές νεοπλασίες

Σκοπός: Οι μυελικές νεοπλασίες είναι κλωνικές διαταραχές του αρχέγονου αιμοποιητικού κυττάρου (ΑΑΚ) και χαρακτηρίζονται από άσκοπο και άμετρο πολλαπλασιασμό ή/και διαταραχή της ωρίμανσης. Οι διαταραχές αυτές χωρίζονται σε δύο μεγάλες ομάδες νοσημάτων, στην Οξεία Μυελογενή Λευχαιμία (ΟΜΛ) και στα Χρόνια Μυελικά Νεοπλάσματα. Η ταξινόμηση αυτή βασίζεται κυρίως στον αριθμό των βλαστών στο περιφερικό αίμα ή τον μυελό των οστών. Με τη σειρά τους, τα Χρόνια Μυελικά Νεοπλάσματα ταξινομούνται σε τρείς κύριες κατηγορίες, τις Μυελοϋπερπλαστικές Νεοπλασίες (ΜΥΝ), τα Μυελοδυσπλαστικά Σύνδρομα (ΜΔΣ) και τα νοσήματα εκείνα που παρουσιάζουν χαρακτηριστικά και των δύο προηγούμενων κατηγοριών (ΜΔΣ/ΜΥΝ) σύμφωνα με κλινικά, μορφολογικά, γενετικά και μοριακά δεδομένα, όπως καταγράφονται στην πρόσφατη αναθεώρηση των διαγνωστικών κριτηρίων που εκδόθηκε πρόσφατα, από τον Παγκόσμιο Οργανισμό Υγείας (Π.Ο.Υ., 2016). Μέχρι πρόσφατα, η διάγνωση και ταξινόμηση αυτών των νοσημάτων, βασιζόταν σχεδόν αποκλειστικά σε κυτταρολογικά χαρακτηριστικά και καρυοτυπικές αναλύσεις, με εξαίρεση πολύ συγκεκριμένες μοριακές βλάβες, όπως εκείνες στα γονίδια JAK2, CALR και MPL, που ανιχνεύονται στην πλειοψηφία των ασθενών με ΜΥΝ, σπανιότερα δε στα ΜΔΣ/ΜΥΝ. Η πρόοδος της τεχνολογίας οδήγησε σε καλύτερο χαρακτηρισμό της παθογένειας των μυελικών νεοπλασιών, με την ανάδειξη πληθώρας μεταλλάξεων σε γονίδια με ετερογενείς κυτταρικές λειτουργίες και μάλιστα με διαφορετική συχνότητα σε κάθε περίπτωση. Χαρακτηριστικό παράδειγμα αποτελούν οι συχνές μεταλλάξεις στα γονίδια JAK2, MPL και CALR, που ανιχνεύονται στην πλειοψηφία των ασθενών με Ιδιοπαθή Θρομβοκυτταραιμία (ΙΘ) ή Μυελοΐνωση (ΜΙ), και οι οποίες θεωρούνται φαινοτυπικές (phenotypic drivers) μεταλλάξεις. Επιπλέον, μεταλλάξεις έχουν αναφερθεί σε γονίδια που εμπλέκονται στη ρύθμιση του επιγενετικού μηχανισμού ρύθμισης του κυττάρου, στον κυτταρικό κύκλο, στην ωρίμανση του mRNA και σε άλλες σημαντικές διεργασίες. Οι μεταλλάξεις αυτές θεωρείται πως διαμορφώνουν τον κλινικό φαινότυπο και συμβάλλουν στην πρόοδο και εξέλιξη της νόσου. Σκοπό της παρούσας Διπλωματικής Εργασίας, αποτέλεσε η μελέτη του Μοριακού τοπίου των ασθενών με μυελικές νεοπλασίες, μέσω στοχευμένου προσδιορισμού της νουκλεοτιδικής ακολουθίας, συχνά μεταλλαγμένων γονιδίων, με χρήση τεχνολογίας δεύτερης γενιάς (targeted panel Next Generation Sequencing) και η ανάπτυξη μίας συμβατής βιοπληροφορικής ροής, για την ανάλυση των δεδομένων. Η διαδικασία αυτή συνεισφέρει στην εύκολη, γρήγορη και αξιόπιστη Μοριακή τυποποίηση των ασθενών με μυελικές νεοπλασίες, συμβάλλοντας έτσι στην ορθή διάγνωση και ταξινόμηση, αλλά και σε πρόγνωση και διαστρωμάτωση του κινδύνου εκτροπής αυτών προς ΟΜΛ, ενώ ιδανικά, δύναται να παρέχει πληροφορίες σχετικά με τις διαθέσιμες θεραπευτικές επιλογές. Μέθοδοι: Στην παρούσα εργασία αναπτύχθηκε μία βιοπληροφορική ροή για την ανάλυση δεδομένων NGS, με στόχο την ανάδειξη μοριακών εξαλλαγών. Για το σκοπό αυτό χρησιμοποιήθηκε η προγραμματιστική γλώσσα Common Workflow Language (CWL) σε συνδυασμό με το λογισμικό Docker, που διευκολύνει τη χρήση, μεταφορά και επαναληψιμότητα της αναλυτικής διαδικασίας. Συνολικά, μελετήθηκαν 51 ασθενείς με μυελικές νεοπλασίες σε Χρόνια ή Οξεία φάση. Αρχικά, απομονώθηκε DNA από περιφερικό αίμα ή μυελό των οστών. Ακολούθησε κατασκευή βιβλιοθηκών DNA, ενώ τέλος πραγματοποιήθηκε στοχευμένος προσδιορισμός της νουκλεοτιδικής ακολουθίας 28 γονίδιων ή μεμονωμένων περιοχών αυτών. Οι περιοχές των γονιδίων που μελετήθηκαν αναφέρονται συχνά μεταλλαγμένα στους ασθενείς με μυελικές νεοπλασίες, όπως υποστηρίζεται από τη σύγχρονη βιβλιογραφία. Αποτελέσματα: Συνολικά αναδείχθηκαν 223 μοριακές εξαλλαγές, εκ των οποίων 29 είχαν ήδη ανιχνευθεί προηγουμένως στο εργαστήριο μας, με ήδη εγκαθιδρυμένες τεχνικές Μοριακής Βιολογίας (post Real-time PCR HRMA και Sanger Sequencing). Από αυτές, 21 μεταλλάξεις εντοπίστηκαν σε 9 ασθενείς με ΜΔΣ, 3 στον μοναδικό ασθενή με ΜΔΣ/ΜΥΝ, 173 σε 33 ασθενείς με ΜΥΝ και 26 στους 8 ασθενείς με ΟΜΛ. Συμπεράσματα: Με την παρούσα εργασία αναδείξαμε τη μεγάλη ετερογένεια του μοριακού τοπίου των μυελικών νεοπλασιών, ακόμα και σε ίδιες ή παρόμοιες κοορτές ασθενών. Παράλληλα εγκαθιδρύσαμε την τεχνική NGS, για την ταυτόχρονη μελέτη πολλών γενομικών περιοχών, ολόκληρων εξωνίων ή και γονιδίων, για περισσότερους από έναν ασθενείς, σε μία μόνο αντίδραση και μάλιστα με υψηλή ευαισθησία. Ταυτόχρονα, προτείνουμε βελτιώσεις, τόσο στο τεχνικό όσο και στο αναλυτικό υπόβαθρο της διαδικασίας, αποσκοπώντας σε βελτίωση της ευαισθησίας και ακρίβειας της μεθοδολογίας, κατά την ανάδειξη μοριακών εξαλλαγών, όπως επίσης σε εμπεριστατωμένη αξιολόγηση της επίπτωσης αυτών στο φαινότυπο της νόσου, με απώτερο στόχο την ενσωμάτωση της τεχνολογίας NGS στην κλινική πράξη.Objective: Myeloid neoplasms are clonal disorders of the hematopoietic stem cell (HSC) and are characterized by excess proliferation and/or disruption of maturation of one or more myeloid cell lineages. These disorders are divided into two major categories, acute myeloid leukemia (AML) and chronic myeloid neoplasms. This classification is mainly based on the number of blasts in peripheral blood or bone marrow. Chronic myeloid neoplasms are classified into three main categories according to clinical, morphological, genetic and molecular data, as reported in the recent revision of the World Health Organization (WHO, 2016), Myeloproliferative Neoplasms (MPNs), Myelodysplastic Syndromes (MDS) and a category with overlapping features of both MDS and MPNs, referred to as Myelodysplastic/Myeloproliferative neoplasms (MDS/MPN). Until recently, the diagnosis and classification of these disorders was based almost exclusively on morphological and cytogenetic features, with the exception of very specific molecular lesions, such as those in the JAK2, CALR and MPL genes, detected in the majority of MPN patients, and more rarely in MDS/MPN. The recent advances in technology have led to a better characterization of the pathogenesis of the myeloid neoplasms, with the identification of an increasing number of genetic defects in genes with heterogeneous cellular functions. A typical example is the frequent mutations in the JAK2, MPL and CALR genes detected in the majority of patients with essential thrombocytosis (ET) and myelofibrosis (MF), which are considered as driver mutations. Additionally, several other mutated genes have been identified and most of these mutations affect a range of essential, interrelated cellular mechanisms such us epigenetic regulation, RNA splicing, transcription and DNA damage response. The various combinations of mutations suggest a multistep pathogenesis and may account for the clinical heterogeneity. The delineation of the complex clonal architectures of these disorders could serve as the cornerstone for better risk stratification of these patients and for the identification of novel therapeutic targets within the context of Precision Medicine. The purpose of this Diploma Thesis was to study the molecular landscape of patients with myeloid neoplasms through targeted re-sequencing, in commonly mutated genes, using Next Generation Sequencing (NGS) technology and developing a portable and user-friendly bioinformatics pipeline for the data analysis. This process enables an affordable, rapid and reliable molecular screening of patients with myeloid neoplasms, contributing thus in a more accurate diagnosis and classification, prognosis and patients’ risk stratification, and could also provide information regarding the available therapeutic options. Methods: In this study a bioinformatics pipeline was developed for the analysis of NGS data aiming at variant calling. For this purpose, the Common Workflow Language (CWL) programming language was used in conjunction with the Docker software, which facilitates the easy use, transfer and repeatability of the analytical process. Overall, 51 patients with myeloid neoplasms in chronic or blast phase were studied. DNA was extracted from peripheral blood or bone marrow. DNA libraries were constructed, and targeted re-sequencing was performed on 28 genomic targets (full genes or specific exons). The selection of the genomic regions under investigation represent mutational hotspots according to recent data and WHO and European LeukemiaNET guidelines. Results: In total, 223 variants were identified, of which 29 were previously detected in our laboratory, with already established methods (Post Real-time PCR HRMA and Sanger Sequencing). Of these, 21 variants were detected in 9 MDS patients, 3 in the only MDS/MPN patient, 173 in 33 MPN patients and 26 in 8 AML patients. Conclusions: In this study, we highlighted the molecular heterogeneity of the genomic landscape of myeloid neoplasms, either within the same or similar cohorts of patients. We also established a custom NGS methodology for the simultaneous analysis of many genomic regions, entire exons and/or genes, for more than one patient, in a single experiment. Furthermore, we propose amendments in both technical and analytical workflow of the process, aiming thus to improve the sensitivity and accuracy of the method regarding variant calling, as well as for the assessment of the impact of these variants on disease phenotype, in order the NGS technology to be reliably incorporated in clinical practice

Archimedes Interdisciplinary Research Programme Forges a Broad Spectrum of Academic Innovations

The basic goal of Archimedes III is to support research teams in Greek Technological Educational Institutes (TEI) and enhance their research capabilities by funding interdisciplinary and inter-institutional research. The programme aims also at attracting talented researchers and developing high quality research personnel. The TEIs, prior to their advancement to the tertiary sector in 2000, focused only on educational activities. Since then, development of research became part of their mission. In order for the Ministry of Education to support TEIsto develop their research capabilities, it designed research programmes targeting exclusively these organisations. Archimedes I and II were designed and ran in the previous programming period (2000-2006) while Archimedes III has been designed in the framework of the Operational Programme "Education and lifelong learning” of the current programming period, 2007-2013. The programme supports research projects in all research fields. However, particular emphasis is given to the fields of engineering and ICT where the main strengths of Greek Technological Educational Institutes are concentrated. The specific project of the TEI of Central Greece, described herewith, comprises 8 research subprojects in fields such as health, environment, energy and ICT with one additional subproject dedicated to management and dissemination. This paper is a comprehensive account of the research objectives and accomplishments of the research project as a whole. This work is licensed under a&nbsp;Creative Commons Attribution-NonCommercial 4.0 International License.</p

Archimedes Interdisciplinary Research Programme Forges a Broad Spectrum of Academic Innovations

The basic goal of Archimedes III is to support research teams in Greek Technological Educational Institutes (TEI) and enhance their research capabilities by funding interdisciplinary and inter-institutional research. The programme aims also at attracting talented researchers and developing high quality research personnel. The TEIs, prior to their advancement to the tertiary sector in 2000, focused only on educational activities. Since then, development of research became part of their mission. In order for the Ministry of Education to support TEIsto develop their research capabilities, it designed research programmes targeting exclusively these organisations. Archimedes I and II were designed and ran in the previous programming period (2000-2006) while Archimedes III has been designed in the framework of the Operational Programme "Education and lifelong learning” of the current programming period, 2007-2013. The programme supports research projects in all research fields. However, particular emphasis is given to the fields of engineering and ICT where the main strengths of Greek Technological Educational Institutes are concentrated. The specific project of the TEI of Central Greece, described herewith, comprises 8 research subprojects in fields such as health, environment, energy and ICT with one additional subproject dedicated to management and dissemination. This paper is a comprehensive account of the research objectives and accomplishments of the research project as a whole. This work is licensed under a&nbsp;Creative Commons Attribution-NonCommercial 4.0 International License.</p

DETECTION OF CALR MUTATIONS USING HIGH RESOLUTION MELTING CURVE ANALYSIS (HRM-A); APPLICATION ON A LARGE COHORT OF GREEK ET AND MF PATIENTS

Background and Objectives Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibosis (MF), lacking the JAK2and MPLmutations. To determine the prevalence of CALRframeshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALRmutants.   Methods MPN patient samples were collected from different clinical units and screened for JAK2and MPLmutations after informed consent was obtained. Negative samples were analyzed for the presence of CALRmutations. To this end, we developed a modified post Real Time PCR High Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing.   Results Using this protocol we screened 173 MPN, JAK2and MPLmutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALRexon 9 mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study reavealed 6 rare novel mutations which are to be added in the COSMIC database.    Conclusions Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups

Role of Histone Deacetylases in the Pathogenesis of Salivary Gland Tumors and Therapeutic Targeting Options

Salivary gland tumors (SGTs) comprise a rare and heterogenous category of benign/malignant neoplasms with progressively increasing knowledge of the molecular mechanisms underpinning their pathogenesis, poor prognosis, and therapeutic treatment efficacy. Emerging data are pointing toward an interplay of genetic and epigenetic factors contributing to their heterogeneity and diverse clinical phenotypes. Post-translational histone modifications such as histone acetylation/deacetylation have been shown to actively participate in the pathobiology of SGTs, further suggesting that histone deacetylating factors (HDACs), selective or pan-HDAC inhibitors (HDACis), might present effective treatment options for these neoplasms. Herein, we describe the molecular and epigenetic mechanisms underlying the pathology of the different types of SGTs, focusing on histone acetylation/deacetylation effects on gene expression as well as the progress of HDACis in SGT therapy and the current status of relevant clinical trials

Bone marrow ribonucleotide reductase mRNA levels and methylation status as prognostic factors in patients with myelodysplastic syndrome treated with 5-Azacytidine

Ribonucleotide Reductase (RNR) is a two-subunit (RRM1, RRM2) enzyme, responsible for the conversion of ribonucleotides to deoxyribonucleotides required for DNA replication. To evaluate RNR as a biomarker of response to 5-azacytidine, we measured RNR mRNA levels by a quantitative real-time PCR in bone marrow samples of 98 patients with myelodysplastic syndrome (MDS) treated with 5-azacytidine with parallel quantification of the gene promoter&apos;s methylation. Patients with low RRM1 levels had a high RRM1 methylation status (p = 0.005) and a better response to treatment with 5-azacytidine (p = 0.019). A next-generation sequencing for genes of interest in MDS was also carried out in a subset of 61 samples. Splicing factor mutations were correlated with lower RRM1 mRNA levels (p = 0.044). Our results suggest that the expression of RNR is correlated with clinical outcomes, thus its expression could be used as a prognostic factor for response to 5-azacytidine and a possible therapeutic target in MDS