Targeted next-generation sequencing for the analysis of somatic mutations in epithelial ovarian cancer
Creators: name: Ryland, Georgina Louise
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Epithelial ovarian cancer is the leading cause of death from gynaecological malignancy, with a poor ratio of survival-to-incidence resulting from the frequent diagnosis of advanced-stage disease where 5-year survival is less than 30%. A limited understanding of the somatic events contributing to ovarian tumorigenesis remains one of the major obstacles to improving treatment outcomes. Recent advances in massively parallel short read or next-generation sequencing (NGS) have provided the greatest opportunity yet for investigating the specific alterations of cancer genomes at single-base resolution. This thesis describes the use of NGS to characterise somatic point mutations and small insertions-deletions (indels) in two ovarian tumour cohorts. As little consensus exists as to the best approach for identifying somatic mutations in paired tumour-germline data, a large component of work focused on establishing robust methodology for this purpose. The specificity of somatic mutation detection was improved by filtering out false positive variants that were contributed to by directionality and misalignment of variant-containing reads, repetitive DNA sequences and incomplete removal of germline polymorphisms. A higher sensitivity was achieved by considering variants called by multiple algorithms, and this strategy particularly helped to improve the detection of somatic indels. The first study using NGS then sought to identify novel ovarian tumour suppressor genes by sequencing genes within common frequent regions of loss of heterozygosity (LOH), hypothesising that these regions will be enriched for classical tumour suppressor genes with somatic mutation as the second hit. Of the 980 genes interrogated, 22 were identified with recurrent mutations characteristic of those which may lead to loss-of-function, although the mutation frequency in all of these genes was low (less than 7%). CASK, GRAMD4 and IL16 were investigated in vitro, however the effects on cell number following gene silencing were inconclusive with respect to tumour suppressive activity. Taken together, the results from this study indicated that regions of LOH are unlikely to harbour any novel frequently mutated classical ovarian tumour suppressor genes. Infrequently mutated genes and other mechanisms of gene suppression, such as haploinsufficiency and epigenetic silencing, should be considered in future studies of tumour suppressor genes in common frequent LOH regions in ovarian cancer. In the second study, whole-exome sequencing was used to characterise somatic mutations in primary mucinous ovarian carcinomas, a rare histological subtype of epithelial carcinomas that has been overlooked in past genetic studies. Combining data from exome sequencing and targeted resequencing identified RNF43 and ELF3 mutations in 21% and 7% of carcinomas respectively. The biallelic inactivating mutations detected in RNF43 were consistent with a role for this RING finger E3 ubiquitin ligase as a tumour suppressor. Furthermore, the mutation data indicated a possible interaction between ELF3, an ETS transcription factor, and ERBB2 amplification. Findings from this study reiterated the importance of RAS/RAF oncogenic activation concurrent with CDKN2A loss in mucinous tumour development and established TP53 mutations as a genetic event promoting an invasive phenotype. Collectively, the results presented in this thesis demonstrate the power of NGS for revealing the genetic complexity of epithelial ovarian cancer.