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ItemThe role of next generation sequencing in the management of haematological malignancies( 2018)Next generation sequencing comprises a rapidly-evolving cohort of technologies which enable detection of genetic variants present in DNA or RNA. In the context of haematological malignancies, such variants may have diagnostic, prognostic or therapeutic relevance. The scope of diagnostic-grade genetic testing is increasing as the underlying molecular landscape of haematological malignancies is increasingly well-characterised and becomes further embedded in clinical management. Multiple conventional methods for the detection of somatic single-gene variants are already embedded in standard-of-care. The technical performance of next generation sequencing when compared to these existing methods is of interest, since this determines in which instances it can be used to supplement or replace the status quo. This work focuses on acute myeloid leukaemia and systemic mastocytosis, since detecting variants in these malignancies presents several technical challenges. Acute myeloid leukaemia demands detection of a broad spectrum of molecular lesions, whereas systemic mastocytosis requires high sensitivity testing. Chapter one reviews molecular testing for haematological malignancies, including aspects of next generation-based testing relevant to diagnostics. Molecular markers currently employed in the diagnosis of acute myeloid leukaemia and systemic mastocytosis are discussed. The second chapter details methods used in the generation of data for chapters three to five. In the third chapter the selection and use of a targeted sequencing panel is discussed, testing performance is compared to conventional molecular methods using data generated from 30 clinical samples. Limitations of panel-based testing are discussed, including sensitivity, detection of specific variant types, and relevant bioinformatic and analytical issues. In chapters four and five, proof of principle is demonstrated for two novel next generation sequencing library generation methods, addressing the issues of structural variant detection ‘HEPTAD’ and high sensitivity single nucleotide variant detection ‘LNA PCRbrary’, respectively. Theory, optimisation and testing results for a combination of 49 clinical samples and cell lines using HEPTAD are discussed. The technique is applied to detect chromosomal translocations involving RUNX1, BCR, ABL, RARA, MLL/KMT2A, a chromosomal inversion involving CFBF, and KMT2A partial tandem duplications. The method detects structural variants in diagnostic samples with 100% sensitivity but requires further optimisation for minimal residual disease testing. Successful prospective application in a clinical scenario where orthogonal FISH testing was performed is detailed. LNA PCRbrary’s application to the detection of KIT D816V single nucleotide variants in peripheral blood is discussed. Suboptimal results when testing RNA derived from clinical samples are detailed, but promising results when testing cell line DNA, and significant potential for further optimisation and testing. In conclusion this work shows that NGS does have a role in the management of haematological malignancies, but technical challenges remain, including high sensitivity detection of single nucleotide variants, minimal residual disease monitoring of structural variants and gene targets which are difficult to amplify. Some of these issues are in part addressed in this work, but careful validation of testing sensitivity and specificity is required when introducing such NGS assays into clinical diagnostics.