Pathology - Theses

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    Genomic alterations in well- and de-differentiated liposarcoma
    Garsed, Dale William ( 2013)
    Well- and de-differentiated liposarcomas (WD/DDLPS) are tumours that arise from fat cells (adipocytes) and are characterised by the presence of supernumerary ring or ‘giant-rod’ marker chromosomes (neochromosomes; NCs). NCs are associated with several cancer types and are thought to harbour oncogenic mutations in the form of amplified oncogenes or fusion genes. However, knowledge of their structure and oncogenic properties is limited. The aim of this thesis is to comprehensively map genomic alterations in WD/DDLPS NCs, and to identify the mechanisms of pathogenesis in this disease. This report describes the first sequence-level map of cancer-associated NCs. A unique targeted genomics approach was developed, in which flow cytometry was used to isolate giant marker NCs from a panel of WD/DDLPS cell lines, and their content and structure was mapped using single nucleotide polymorphism mapping arrays and next-generation sequencing analyses. The NCs have a complex spatial architecture, with over 900 structural variations, the majority of which were formed by DNA translocations showing evidence of nonhomologous end-joining repair. I developed a novel classification system defining distinct patterns of breakpoints and fusions, which formed the basis for detailed analysis of this complex structure. In-depth characterisation of DNA fusions revealed the molecular history of NC evolution. Different patterns of rearrangement were observed, including evidence of localised shattering and random repair (chromothripsis) on the long arm of chromosome 12, which may be a fundamental mechanism in NC formation. Following initial assembly, DNA amplification occurs by breakage-fusion-bridge cycles in the ring conformation, a phase terminated by linearization and the acquisition of functional telomeres from normal chromosomal counterparts. A genome-wide survey of copy number variations (CNVs) was carried out in primary WD/DDLPS, to define regions most likely to harbour oncogenes and tumour suppressors. The most significant regions of CNV were determined by measuring the frequency of occurrence and amplitude of DNA gain or loss. The genes identified in these candidate regions were then triaged to a list of candidate genes suitable for functional validation, by (i) testing for copy number correlation with matched mRNA expression data, (ii) identifying biologically significant genes based on the literature, and (iii) comparing these findings to a previously published independent data set. A number of novel copy number alterations were identified, including amplification of NUP107, OS9 and CYP27B1, and deletion of DDX6, HRAS, and OTUB1. In order to refine the list of candidate oncogenes, a functional validation screen was performed using RNA interference (RNAi) to determine which amplified genes are necessary for WD/DDLPS cell growth. Short interfering RNA (siRNA)-mediated depletion of several amplified genes was anti-proliferative to WD/DDLPS cells, including the nucleoporin NUP107, recently reported to be over-expressed in some cancers. NUP107 is amplified in 53% of primary WD/DDLPS tumours, and its attenuation caused growth suppression and cell cycle arrest in vitro. In addition to copy number changes driving over-expression, transcriptome sequencing revealed alterations in mRNA splicing, cryptic exon usage, and fusion transcripts, presumably as a result of NC rearrangements. Alternatively spliced transcripts of the oncogene MDM2 are specifically expressed in WD/DDLPS tumours and are expressed at a low level or absent in normal fat tissue and other tumour types. Preliminary data indicates that the expression of altered mRNA may have a role in carcinogenesis. Taken together, the findings in this thesis provide exciting new insights into the structural complexity, evolution and pathogenesis of WD/DDLPS NCs.