Study of molecular abnormalities in patients with myeloid malignancies utilizing NGS targeted re-sequencing

Postgraduate Thesis uoadl:2879608 436 Read counter

Unit:
Κατεύθυνση Βιοπληροφορική-Υπολογιστική Βιολογία
Library of the School of Science
Deposit date:
2019-07-23
Year:
2019
Author:
Loupis Theodoros
Supervisors info:
Καθηγητής Ιωάννης Π. Τρουγκάκος, Τομέας Βιολογίας Κυττάρου & Βιοφυσικής, Τμήμα Βιολογίας, ΕΚΠΑ
Επίκουρη Καθηγήτρια Βασιλική A. Οικονομίδου, Τομέας Βιολογίας Κυττάρου & Βιοφυσικής, Τμήμα Βιολογίας, ΕΚΠΑ
Επίκουρος Καθηγητής Χρήστος Κοντός, Τομέας Βιοχημείας & Μοριακής Βιολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Original Title:
Στοχευμένος προσδιορισμός αλληλουχίας, με τεχνολογία δεύτερης γενιάς (Next Generation Sequencing), σε ασθενείς με μυελικές νεοπλασίες
Languages:
Greek
Translated title:
Study of molecular abnormalities in patients with myeloid malignancies utilizing NGS targeted re-sequencing
Summary:
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.
Main subject category:
Science
Keywords:
Myeloid Neoplasms, Next Generation Sequencing, CWL, Docker
Index:
Yes
Number of index pages:
2
Contains images:
Yes
Number of references:
130
Number of pages:
105
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