Pathology - Theses
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ItemImage guided and adaptive radiotherapy for muscle invasive bladder cancer( 2014)Introduction: Bladder cancer is one of the ten most frequent cancers in Australia. It is also the only common cancer for which survival has decreased over the last twenty years. The two curative treatment options for muscle invasive bladder cancer are radical surgery requiring removal of the bladder, or radical radiotherapy (alone or in combination with chemotherapy). Radical radiotherapy allows many patients to keep their natural bladder. As a dynamic soft tissue organ, the bladder size, shape and position vary with bladder and rectal filling, requiring traditional radiotherapy fields to have large margins around the target. With conventional radiation treatment such large margins increase the risk of normal tissue side effects and yet there still remains the risk of missing the bladder cancer on some treatment fractions. Methods: I have developed an innovative technique with a new device, cone beam computed tomography to match radiation fields and volume on a daily basis to the bladder position and size. This technique reduced the margin of the radiation fields required around the bladder. I have led a number of training programs and their evaluation to teach radiation therapists to conduct such treatments. In addition to the development work, I have conducted a prospective pilot study in 27 participants, of this adaptive radiotherapy technique. Following further refinement I led the multi-centre clinical trial that established the technique as standard of care in a number of institutions. Results: I found that cone beam computer tomography was of sufficient quality to be used to match radiation fields to the bladder on a daily basis. I determined that such daily matching prior to treatment was better than an ‘offline” process where an average radiation treatment plan was created after several radiation treatments. I found in our pilot study that adaptive radiation treatment decreased surrounding normal tissue irradiation. Separate studies showed that both workshop and e-Learning based radiation therapist training increased confidence and decreased variation from the gold standard (radiation oncologist results). I conducted a number of studies examining appropriate margins for bladder cancer radiation treatment with different imaging techniques. Through a multi-centre feasibility study of 54 participants, I introduced the image guided adaptive radiotherapy technique into a number of Australia centres, and demonstrated that, while technically possible, the margin around the bladder in this protocol was too small. Conclusions: Image guided and adaptive radiotherapy is possible in many radiation therapy departments with likely benefits for patients in terms of cancer control and reduced normal tissue side effects. Through my work the technique has been established as standard of care in a number of Australian and New Zealand centres. However such radiotherapy techniques can continue to be optimised. Future phase III studies are required to conclusively prove their benefit.
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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.
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ItemOcular pathology: a compilation of research articles( 2011)These articles comprise publications in the field of ocular pathology over 15 years, with particular interest in lymphomas of ocular adnexal and intra-ocular sites; ocular impression cytology and its application in the diagnosis and management of ocular surface squamous neoplasia; extraocular muscle in ageing and disease; diode laser photocoagulation of ciliary body in intractable glaucoma; rare and unusual ocular infections and tumours and early giant cell arteritis.
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ItemMolecular markers in sporadic and BRCA1-associated basal breast cancers( 2011)Basal breast cancers are a subgroup of breast cancers with a distinctive gene expression profile, basal cytokeratin expression, and a “triple negative” phenotype (i.e. negativity for hormone receptors and HER2 amplification). There are currently limited options for targeted therapy in these cancers. The aim of this thesis is to characterise some of the critical pathways that are responsible for the pathogenesis of these breast cancers and to identify molecular markers that may predict prognosis and response to treatment. MicroRNAs (miRNAs) are small non-coding RNAs that suppress translation through imperfect base pairing with target mRNAs. There are limited data regarding miRNA expression in basal cancers and its impact on protein synthesis. I hypothesised that i) basal breast cancers have a distinctive miRNA profile, and ii) differences in miRNA profiles exist between sporadic and BRCA1 basal cancers. Analysis of miRNA array data generated from 44 grade 3 cancers (including 11 BRCA1 basal, 16 sporadic basal and 17 luminal cancers) revealed a distinctive “basal” miRNA signature. Via a combination of target prediction algorithms and immunohistochemistry, it was shown that in basal cancers, miRNAs may be involved in the regulation of important regulatory pathways such as the MAPK/ERK pathway. It was also shown that BRCA1 and sporadic basal cancers differ in their miRNA profiles. This was reflected by differences in the expression of genes such as cyclin D1, NRP1 and FOXP1. These findings may have important diagnostic implications, as immunohistochemical assessment of basal cancers, in addition to a detailed family history, may potentially identify patients who may benefit from BRCA1 mutational analysis. I also hypothesised that prognosis and response to treatment in basal cancers may be affected by factors in the tumour microenvironment, such as hypoxia and the anti-tumour immune response, and by intrinsic cellular mechanisms such as cohesin/Rad21 mediated DNA repair and ERβ expression. Investigation of molecular marker expression in cohorts of sporadic and familial breast cancers revealed the behaviour of basal cancers is partly due to an enhanced hypoxic response. In BRCA1 basal cancers, enhanced hypoxic drive may be a consequence of suppressed PHD activity and aberrantly located FIH expression. This hypoxic response may also drive regulatory T cell (Treg) recruitment by inducing CXCR4 up-regulation in Treg, resulting in the suppression of the anti-tumour immune response. Lastly, hypoxia, cohesin/Rad21 mediated DNA repair via homologous recombination (HR) and cytoplasmic ERβ2 expression may impair the response of basal cancers to chemotherapeutic agents. For cohesin/Rad21, this was supported by in vitro experiments where Rad21 knockdown in a basal cell line was associated with enhanced chemosensitivity to DNA damaging agents. In summary, the findings in this thesis highlight the potential diagnostic, prognostic and predictive roles of a number of molecular markers in basal breast cancers. In the era of molecular medicine, these findings support the potential use of miRNAs as a diagnostic marker of BRCA1 status, and the development of therapy directed against specific pathways (i.e. hypoxia, regulatory T cells and HR repair) in these aggressive cancers, for which there is currently no targeted treatment available.