Pathology - Theses
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ItemInvestigation of clustered hypermutation in cutaneous melanoma( 2017)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.
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ItemInvestigating the predictors of sensitivity to the Chk1 inhibitor PF-00477736 in melanoma( 2013)Chk1 and Chk2 are two of the most important and highly studied checkpoint kinases that help protect cells from endogenous or exogenous insults which could damage DNA. When activated they arrest cell cycle at specific checkpoints in order for cells to repair damage before resuming the cell cycle and mitosis. Recently, several inhibitors of Checkpoint kinases have been developed which were designed to sensitise TP53 mutant cancers with defective G1 checkpoints to DNA damaging chemotherapy. However, a recent study has demonstrated that TP53 mutation alone does not predict sensitivity to Chk1 inhibitors. Moreover, inhibition of Chk1 by PF-00477736 (PF-736) as a single agent was also recently shown to be effective in mediating p53-dependent apoptosis in lymphoma cells by increasing the level of MYC oncogene-induced DNA damage. Other reports have also shown that mutant BRAF and RAS (NRAS) which are the predominant oncogenes found in melanoma could induce DNA damage in epithelial cells and thyroid cancers, respectively. This led to the hypothesis that melanomas which are predominantly TP53 wild-type and BRAF or NRAS mutant would be responsive to Chk1 inhibitors as single agents. Screening for drug sensitivity across a panel of 30 melanoma cell lines showed a wide range of IC50 values following 3 days treatment with PF-736. BRAF and TP53 mutation status were not found to affect sensitivity to PF-736. Due to unavailability of NRAS mutation data at the time of analysis, it is still unconfirmed whether BRAF and NRAS as the source of oncogene DNA damage in melanoma influence the sensitivity of melanoma to PF-736 treatment. Several cell lines categorised as sensitive was revealed to have high Total Growth Inhibition (TGI) values. This suggests that there may be genetic variations in the cells that have high TGI particularly in cell death activation pathways that affect the sensitivity of these cells to Chk1 inhibition. Previously, it has been reported that up-regulation of p53 negative-regulators such as Mdm4 and Bcl-2 family anti-apoptotic proteins were responsible for the defective p53-mediated apoptosis responses in p53 wild-type melanomas. Overexpression of BCL2 or BCLxl was able to prevent apoptosis in C002-M1, a cell line that underwent apoptosis following PF-736 treatment. However, MCL1 overexpression did not result in similar effect. Assessment of Mdm4 basal protein levels did not reveal any significant correlation with PF-736 IC50. Further assessment utilising MDM4 knockdown by shRNA in the A375 cell line expressing high Mdm4 protein expression did not result in sensitisation to PF-736. These data suggested that the increased levels of Bcl-2 or Bcl-XL proteins were able to decrease the level of apoptosis but did not significantly alter sensitivity to PF-736 in C002-M1 cell line. Additionally knockdown of MDM4 levels did not alter PF-736 IC50 in a cell line with high Mdm4 protein expression. Hence it is possible that other cellular characteristics such as the level of inherent DNA damage recently reported to be important in sensitivity of melanoma to Checkpoint kinase inhibition, could provide more insight in revealing factors that determine the sensitivity of PF-736 in melanoma. γH2AX is a marker of double strand break which can be utilised as a measure of DNA damage. In an attempt to assess γH2AX as a biomarker of sensitivity, the levels of inherent γH2AX and fold change following PF-736 treatment was determined. The result showed that high level of inherent γH2AX showed a significant correlation with low PF-736 IC50, suggesting that high levels of endogenous DNA damage is associated with sensitivity to PF-736. Furthermore, the average fold increase in levels of γH2AX after PF-736 treatment across the entire cell line panel also significantly correlated with PF-736 IC50. Additionally, we discovered that increase in pChk2 T68 levels following PF-736 treatment also correlated with response to PF-736 in the 30 melanoma cell line panel. Increase in pChk1 S345 following Chk1 inhibition, previously reported as a potential pharmacodynamic biomarker of Chk1 inhibition was confirmed across the panel following the same PF-736 treatment demonstrating the biomarkers identified were associated with Chk1 inhibition. A significant component of this project was to identify predictors of sensitivity using a pharmacogenomic whole genome-wide approach and a candidate approach. For the candidate approach, gene expression of selected genes from pathways related to the DNA damage response pathway for the 30 melanoma cell line panel were analysed with IC50 from SRB assay utilising linear regression analysis to search for significant correlation between sensitivity to PF-736 and gene expression. Gene expression of NBN, CHK2, RAD21 and RAD54B, which are components of the DSB repair particularly HR repair, significantly associated with sensitivity to PF-736. An unbiased Pearson correlation analysis between whole-genome gene expression microarray data and IC50 data from SRB assay of the 30 melanoma cell line panel also showed good correlation between DSB repair genes and sensitivity to PF-736. From this analysis, RAD21 and RAD54B were showed to have a good correlation with sensitivity to PF-736. An SVM prediction model built using the gene expression data pre-processed using the Pearson correlation analysis to include only genes that are differentially expressed across the panel and their sensitivity to PF-736 based on IC50, to discover a gene set that can classify melanoma cell lines between sensitive and resistant to PF-736. The result showed that the SVM model was able to predict at 100% accuracy with a cut-off of 2048 nM. Some of the genes included in the gene set for the SVM prediction model were genes that are important for DSB repair particularly HR such as RAD21, RAD54B and NSMCE2 confirming the importance of this specific DNA repair pathway in association with sensitivity to PF-736 particularly in melanoma. In conclusion, this study has revealed the importance of the DSB Response pathway in predicting the sensitivity to the Chk1 specific inhibitor PF-736 in melanoma. As there are also several other cancers which exhibit oncogene-induced DNA damage and aberrant DSB repair pathways, this study may help in predicting the sensitivity of these types of cancers in with single agent Chk1 therapy. Furthermore, this project has verified that melanoma cell lines are sensitive to Chk1 inhibition therapy as a single agent and that the level of inherent DNA damage may provide insight into the sensitivity of melanoma to Chk1 inhibition. Moreover, candidate and genome-wide pharmacogenomic analyses have revealed the importance of DSB repair pathway and Bcl-2 family anti-apoptotic genes in predicting sensitivity to Chk1 inhibition. Although functional study on C002-M1 cell line did not show evidence that the level of Bcl-2 family anti-apoptotic proteins can affect sensitivity, the western blot data, candidate gene and genome wide predictor analysis across the 30 melanoma cell line panel showed that there is a significant correlation between Bcl-2 family anti-apoptotic proteins particularly Bcl-XL to sensitivity to PF-736, which warrants further investigation. As a future direction, the current predictor model will need to be tested with a validation set of samples, to test its robustness in predicting sensitivity to single agent Chk1 inhibition therapy. To progress further the assessment of the usefulness of Chk1 inhibition therapy in melanoma, an investigation on Chk1 inhibition in combination with DNA damaging chemotherapy which was shown to be promising in the preliminary study, will need to be carried out.
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ItemClinical implications of melanin expression in melanoma and molecular drivers of cutaneous squamous cell tumours( 2013)Melanomas are derived from melanocytes – highly specialized pigment-producing cells residing in the basal layers of the skin. The majority of melanomas continue to produce melanin, presenting a potential disease specific biomarker, so far largely unexplored. This thesis begins by examining how melanin might be exploited as a melanoma specific target for molecular based imaging. The novel fluorinated, melanin-binding radiopharmaceutical, MEL050, is tested as a PET-tracer in ten patients with advanced melanoma. We establish MEL050 safety, and detail metabolism, radiopharmacokinetics and normal biodistribution. Diagnostic parameters are compared with a gold-standard of FDG PET/CT. Excellent specificity of 100% but more variable sensitivity is demonstrated. Overall sensitivity was 47%. In a subset of five patients who underwent resection of all known disease sites, histological assessment of tumour melanin correlated with MEL050 PET signal: 100% for melanotic tumours but 0% for amelanotic tumours. These findings suggested a high frequency of amelanotic metastases – an aspect of melanoma not well detailed in the literature. In order to validate melanin as a suitable target for melanoma imaging, but also to determine if changes in its expression may reflect clinically meaningful differences in underlying biology, we next examined melanoma melanin in two distinct clinical cohorts. In the first, amelanosis was evaluated in tumours from 253 patients with primary melanoma. 20% were macroscopically amelanotic. These tumours had a higher Breslow thickness (p < 0.001), were more likely to be ulcerated (p = 0.002), have increased mitoses (p < 0.001) and be BRAF wild-type (p < 0.001). In a multivariate survival analysis, amelanosis was associated with worse disease free survival (HR 2.3, p = 0.031) and disease specific survival (HR 2.5, p = 0.033). The second cohort examined patients with more advanced melanoma. 142 patients with stage IIIa melanoma had sentinel node tumours examined for novel markers of melanin and Ki-67. 45% of tumours were amelanotic by H&E and mean Ki-67 score was 29%. Sentinel node tumour melanin outperformed existing prognostic factors, however, contrary to in primary tumours, persistent melanin was associated with worse disease free (HR 2.1, p = 0.002) and disease specific survival (HR 3.6, p = 0.009). Furthermore, in matched biopsies from primary, regional and distant metastatic sites, the frequency of amelanosis was shown to increase with disease progression – although bidirectional phenotype switching was also observed. Finally, an interaction between molecular targeted therapies and cutaneous squamous cells in a subset of melanoma patients was explored. Using mass-spectrometric genotyping, mutations in cutaneous squamous cell tumours developing in RAF-inhibitor treated patients were compared with similar tumours developing spontaneously or in patients requiring immunosuppression. Treatment related tumours were enriched for RAS mutations (OR 8.0, p = 0.007) supporting a hypothesized proliferative interaction between RAF-inhibitors and RAS primed cells. Amalgamated findings suggest dynamic changes in melanogenesis make melanin an unsuitable target for diagnostic imaging. Clinicopathological and prognostic associations of amelanosis in the context of known regulatory pathways support a distinct phenotype worthy of further clinical exploration and biological characterization. Confirmation of a clinical interaction between RAF inhibitors and RAS activated cells provides strategies for anticipating and managing treatment related toxicities.