Pathology - Theses
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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.
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ItemThe genomic landscape of phaeochromocytoma( 2015)Phaeochromocytomas (PCC) and paragangliomas (PGL) (collectively PPGL) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PPGL cases are associated with heritable syndromes involving 18 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PPGL, exome sequencing, high-density SNP-array analysis, and RNA sequencing was applied to 36 PCCs and four PGL tumours. All tumours displayed a low mutation frequency in combination with frequent large segmental copy-number alterations and aneuploidy, with evidence for chromothripsis seen in a single case. Thirty-one of forty (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PPGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation (e.g. TP53, STAG2, KMT2D). A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function (e.g. ASCL1, NCAM1, GOLGA1). In addition, the existing paradigm for gene-expression subtyping of PPGL was further refined by applying consensus clustering to a compendium of previously published microarray data, enabling the identification of six robust gene-expression subtypes and subsequent cross-platform classification of RNA-seq data. The majority of cases in the cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PPGL, suggesting there are yet unknown PPGL cancer genes that can phenocopy MAX mutations. The cross-platform classification model was then further refined to develop a 46-gene Nanostring-based diagnostic tool capable of classifying PPGL tumours into gene-expression subtypes. The strong genotype-to-subtype relationship in PPGL makes subtyping a powerful tool that can be used clinically to guide and interpret genetic testing, determine surveillance programs and aid in better elucidation of PPGL biology. In applying the diagnostic assay to a test set of 38 cases, correct classification into one of the six subtypes was achieved for 34 (90%) samples based on the known genotype to gene-expression subtype association. The observation that at least one of the six subtypes is likely defined by the presence of non-neoplastic cells led to further refinement into five, four, and three-class architectures, further improving classification accuracy. Increasingly tumour heterogeneity is being recognised as one of the most significant challenges facing modern oncology. Genomically diverse tumour regions create additional complexity in predicting treatment response and metastatic potential through biopsy. Multi-region sampling of multiple synchronous primaries from patients with a predisposing germline mutation was used to explore tumour evolution and heterogeneity in PPGL and concomitant medullary thyroid carcinoma. Evolutionary reconstruction of a single primary PPGL demonstrated periods of both branched and linear evolution resulting in a high degree of intratumoural heterogeneity. Comparison of multiple synchronous primaries provided strong evidence of convergent evolution through recurrent chromosomal aberrations, indicating these may be obligate events in tumourigenesis, and as such, may indicate potential novel therapeutic targets.