Investigation of clustered hypermutation in cutaneous melanoma
AuthorColebatch, Andrew James
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-02-02. This item is currently available to University of Melbourne staff and students only, login required.
© 2017 Dr. Andrew James Colebatch
Cutaneous melanoma is a malignant neoplasm that arises from melanocytes in the skin, and is a significant cause of morbidity and mortality, especially in Australia. Next generation sequencing studies of cutaneous melanoma have confirmed the role of UV mutagenesis in its aetiology and demonstrated the high somatic mutation burden in this disease. To date, the majority of genomic analyses of cutaneous melanoma have limited their evaluation of somatic mutations to protein coding regions in order to detect cancer driver mutations. The recent discovery of driver mutations within the TERT promoter however has shown that biologically significant somatic events occur within non-coding regions of the melanoma genome. This thesis set out to further explore the presence, timing and biology of promoter-based somatic mutations in cutaneous melanoma. In this thesis, a systematic analysis of mutations in cutaneous melanoma demonstrated enrichment of somatic mutations within active promoters. Furthermore, somatic mutations were located within sequence motifs matching binding sites for ETS and Sp1 transcription factors. The number of promoter mutations within a tumour correlated with clinicopathological markers of chronic UV exposure and overall somatic mutation load. Strikingly, the same promoter mutations were detected in other cutaneous malignancies. In order to evaluate the potential of promoter mutations as biomarkers, a highly sensitive assay was developed to detect TERT promoter mutations, the most prevalent and biologically plausible promoter mutation. Using this assay on a series of benign melanocytic naevi identified TERT promoter mutations in 2/17 samples, providing evidence that these driver mutations occur in benign neoplasms with implications for the interpretation of TERT mutations in borderline clinical samples. Microdissection of melanomas with coexistent radial and vertical growth phases demonstrated that a majority of promoter mutations are early events in melanomagenesis. TERT promoter mutations are shown to sometimes occur late in melanoma development and show coexistent subclones. In addition, somatic promoter mutations were detected in non-neoplastic skin adjacent to melanoma. Finally, a somatic mutation load assay was designed and evaluated on clinical melanoma samples. An analysis of publically available melanoma exome data demonstrated a subgroup of highly mutated BRAF/NRAS wildtype cutaneous melanomas. The assay was tested on clinical samples and was shown to be incapable of accurate determination of mutation load in melanoma due to technical limitations. In conclusion, the results of this thesis demonstrate a novel somatic mutational process at work in cutaneous melanoma represented by hypermutation within specific promoters, and evaluates these, along with the previously described TERT promoter mutation, as potential biomarkers. In so doing, these findings contribute to understanding of the biology, aetiology and role of somatic promoter mutations, critical knowledge for their potential future use in clinical assays.
Keywordsmelanoma; genomics; ultraviolet radiation; noncoding mutations
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- Pathology - Theses