Date of Award

2021

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical & Health Sciences

First Advisor

Associate Professor Elin S. Gray

Second Advisor

Dr Leslie Calapre

Third Advisor

Professor Mel Ziman

Abstract

Metastatic melanoma is responsible for almost 80% of all skin cancer-related deaths and the incidence of people affected continues to rise worldwide. The emergence of targeted therapy and immune-checkpoint inhibitors has improved the clinical management of melanoma, but durable survival benefit is only seen in a minority of patients. The use of these very expensive systemic therapies on all appropriate patients also poses a high economic burden on health systems across numerous countries. Currently, surveillance for treatment failure is not optimal. Thus, reliable and accurate biomarkers of patient disease status are urgently required.

Circulating tumour DNA (ctDNA) analysis has emerged as a potential “liquid biopsy” for melanoma. Plasma-derived ctDNA are short DNA fragments released into the bloodstream by apoptotic tumour cells. Studies have shown that ctDNA levels in blood correlate with tumour burden and can comprehensively capture the molecular heterogeneity of melanoma metastases. Thus, ctDNA appears to be a viable biomarker for monitoring treatment response and disease progression in melanoma patients. However, further studies aimed at comparing ctDNA and current standard clinical assessments are needed to fully define its suitability as a complementary test to guide treatment decisions.

This thesis aims to provide important information that will assist with the implementation of ctDNA as a biomarker for melanoma in the clinical management of the disease. This thesis is comprised of 7 chapters: a comprehensive literature review (Chapter 1. Introduction); a materials and methods chapter (Chapter 2); 4 results chapters (Chapter 3 – 6); and a final chapter with a general discussion of main findings and future directions (Chapter 7. General Discussion and Future Directions).

The first chapter of the thesis includes a thorough review of the literature on ctDNA as a potential biomarker for melanoma disease (Chapter 1). This is then followed by a detailed description of our protocol for plasma ctDNA extraction and quantification using droplet digital PCR (Chapter 2). Using this methodology, we evaluated the ctDNA detection rate in untreated BRAF mutant melanoma patients, as a potential alternative to tumour genotyping (Chapter 3), where the potential economic benefit of implementing plasma ctDNA testing by ddPCR relative to tissue BRAF testing was also investigated.

The study in Chapter 4 demonstrated that pre-treatment plasma ctDNA is predictive of patient outcomes in the first-line treatment setting. However, baseline ctDNA level was not predictive of outcomes in the second-line immunotherapy setting, especially in patients that were pre-treated with BRAF/MEK inhibitors. Moreover, we found preliminary evidence that patients with high pre-treatment ctDNA may benefit from combined anti-CTLA-4/anti-PD-1 therapy.

Chapter 5 discusses the validity of ctDNA as a surveillance biomarker for melanoma. The kinetics of ctDNA decline were found delayed in patients treated with immunotherapy compared to those receiving MAPK inhibitors. Nonetheless, decreasing ctDNA levels within 12 weeks of immunotherapy or BRAF/MEK inhibitors was strongly concordant with treatment response and significantly associated with longer progression-free survival (PFS). Furthermore, exploratory analysis of nine patients commencing anti-PD- 1 therapy showed a trend of high tumour mutational burden (TMB) and neoepitope load in responders compared to non-responders.

Chapter 6 evaluates the validity of ctDNA to accurately detect disease progression using both a retrospective and a prospective cohort of melanoma patients. The results indicated a moderate detection rate, suggesting that more sensitive methodologies are required to achieve a limit of detection comparable to current medical imaging.

Finally, Chapter 7 provides a general discussion of the studies covered in this thesis. It underscores the clinical validity of ctDNA as a biomarker of prognosis and therapeutic response in melanoma patients, while highlighting important limitations inherent to ctDNA analysis that need to be thoroughly addressed before it can be successfully implemented in the clinic.

Access Note

Chapters 2, 3 and 6 are not available in this version of the thesis, as well as some images from Chapter 1.

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