Systematic longitudinal analysis of circulating tumour DNA in melanoma patients undergoing systemic therapy

Background: Recent advances in targeted and immunotherapies have unlocked potent treatment options for malignant melanoma patients. However, targeted therapies are associated with limited response durations and only a subset of patients achieves durable disease control on immunotherapies. The analysis of circulating tumour DNA (ctDNA) provides a powerful tool for the continuous assessment of treatment responses. Material and Methods: We have established a robust platform for the analysis of ctDNA, using a combination of next generation sequencing (NGS) and droplet digital PCR (ddPCR) based approaches. Results and Discussion: For routine patient follow-up, first pre-treatment DNA isolated from biopsy material or plasma was tested to confirm mutation status and rule out pre-existing resistance-associated mutations using a custom NGS panel. Timely follow-up throughout treatment was then achieved by quantifying ctDNA levels using mutation-specific ddPCR assays. For patients on therapies targeting mutant BRAF, additional ddPCR-based profiling for a panel of NRAS mutations and NGS-based re-sequencing for other resistance-associated mutations was initiated once increasing ctDNA levels were detected. We demonstrate superiority of ctDNA monitoring over LDH measurements and present a strategy to detect treatment failure or emerging resistant disease ahead of clinical scans in a cost-effective, timesaving and low technology way. In addition, we have analysed ctDNA to uncover clonally distinct tumour sub-populations resulting in complex responses to treatment. We monitored a case of metastatic vaginal mucosal melanoma undergoing sequential targeted, immuno- and chemotherapy. Despite the presence of a KIT mutation in the primary tumour, response to the KIT inhibitor imatinib was mixed. Whole exome sequencing of ctDNA followed by targeted longitudinal follow-up revealed a KIT-mutant tumour sub-clone that responded to imatinib and a second sub-clone presenting with an SF3B1 mutation that did not respond. The sub-clones also responded differentially to immunotherapies, but both responded to chemotherapy. Conclusion: The analysis of ctDNA is a powerful approach to monitor responses to systemic therapies in melanoma. By providing early warning of disease progression and crucial information of clonal composition/evolution of the disease, ctDNA analysis has the potential to revolutionise the implementation of precision medicine.