Document Type

Theses, Ph.D


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Publication Details

Thesis successfully submitted for the award of PhD, Technological University Dublin.


The last decade has seen significant development in understanding the genetic basis of myeloproliferative neoplasms (MPN), with the underlying somatic mutational landscape revealed by genome and targeted exome sequencing. The aim of this research was to conduct a series of original investigations to develop insight into the utility and clinical application of these disease markers. Initially, three recently identified disease-specific molecular markers (mutations in JAK2, CALR and CSF3R) were utilised to measure the burden of disease post allogeneic transplantation using methods based on quantitative PCR, capillary electrophoresis and Sanger sequencing. The measured variant allelic frequency of the disease was compared against percentage donor chimerism – a short tandem repeat PCR based method with high discriminatory power that enables a semi-quantitative measure of engraftment based on the calculation of donor and recipient DNA in the blood. All three mutation markers were shown to be stable and robust for monitoring purposes throughout the disease course, inversely correlating with percentage donor chimerism. The increased sensitivity of the JAK2 V617F qPCR used in the JAK2 study highlighted the potential of these disease specific markers to detect molecular and haematological relapse in patients several weeks before clinical manifestation and proved an advantageous adjunct in the clinical assessment of transplantation efficacy. In a second series of studies, the incidence and significance of recurrent MPN mutations in specific clinical scenarios and MPN disease cohorts was investigated. A baseline for the incidence of JAK2, CALR and MPL mutations in Irish patients referred for suspected MPN was established and shown to be comparable with that of other Western European cohorts. To extend this finding, the incidence and significance of these mutations was investigated in specific clinically relevant disease cohorts, revealing a distinct molecular IV pathogenesis in paediatric patients with essential thrombocythaemia (ET) when compared to adults, a low incidence of CALR mutations in splanchnic vein thrombosis patients that correlated with the aetiology of the disease and the presence of CALR driver mutations in familial MPN cases. Additionally, the presence of a JAK2 V617F mutation in utero in ET was established for the first time and the dynamic nature of MPN disease clones over the course of disease was revealed, thus informing insight into the clonal evolution and molecular heterogeneity of MPN patients. This information was collated with results from a number of audits based on clinical referral patterns to a routine molecular diagnostic facility to inform the development of a molecular diagnostic testing algorithm for patients with suspected MPN. In the third series of studies novel next generation sequencing (NGS) technology was applied to investigate somatic mutations in acute myeloid leukaemia (AML) related patient cohorts. A proprietary NGS panel for recurrently mutated genes possessing diagnostic and prognostic significance in AML was evaluated on the Ion Torrent (ThermoFisher) NGS platform across three molecular diagnostic centres in Ireland, Northern Ireland and Scotland. This verified the technique to be robust and reproducible for mutation detection in AML patients. The technology was subsequently deployed to profile mutation status in a clinically high-risk AML patient cohort undergoing transplantation therapy and in a familial AML disease setting to clarify the genotype, reveal the diverse clonal architecture of patients and identify additional driver mutations relevant for existing and emerging therapeutic interventions. Taken together, the studies in this thesis inform clinical understanding and decision making in MPN and AML related settings, provide novel insight into underlying mutational status and clonal behaviour in myeloid malignancies and have the potential to translate into optimised and improved treatment for these patient cohorts.