Childs, Alexa; (2019) The Molecular Characterisation of Circulating Tumour Cells in Neuroendocrine Neoplasms. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Identification of the molecular alterations that drive cancer is critical for precision oncology. Profiling of a single tissue biopsy is insufficient to interrogate the full spectrum of molecular heterogeneity that exists within a patient’s tumour, and is not without risk to the patient. The analysis of CTCs as part of a liquid biopsy circumvents this issue and allows single-cell analysis as well as longitudinal monitoring over time and in response to therapy. The aim of this thesis is to perform the first molecular characterisation of CTCs derived from NEN patients with a view to evaluating therapeutic targets and characterising tumour heterogeneity and evolution at the single-cell level. Firstly, I developed an assay to enable detection of the therapeutic targets SSTR2 and SSTR5 on individual NEN CTCs. Applied to a cohort of 31 metastatic NEN patients, I identified an SSTR+ subpopulation in 33% of patients and demonstrated significant intra- and inter-patient heterogeneity of SSTR expression. Next, I evaluated the size-based Parsortix platform for CTC enrichment against the gold standard EpCAM-dependent CellSearch in a pilot study of NEN patients, demonstrating that a higher number of CTCs could be isolated in a greater proportion of NEN patients using this technique. Furthermore, the presence of CTCs with low and absent EpCAM expression was observed for the first time in NEN alongside significant intra-patient heterogeneity in EpCAM expression. In order to fully dissect the heterogeneity observed in this early work, I developed DEPArraybased workflows to allow the single-cell evaluation of CTC copy number profiles using next-generation sequencing. The developed methodologies were subsequently tested in a representative cohort of NEN patients. By performing comprehensive copy number profiling of 125 single CTCs, I was able to identify recurrent and therapeutically relevant rearrangements, such as the amplification of CDK4/6, MET and BRAF and loss of BRCA2. Unsupervised hierarchical clustering demonstrated CTCs with distinct clonal lineages and significant heterogeneity was seen in CNV profiles between and within patient samples. In conclusion, this thesis describes successful workflows for the genomic analysis of CTCs at the single-cell level and is a step towards the implementation of precision oncology in neuroendocrine patients. This analysis has identified CTC heterogeneity at the single-cell level with implications for the identification of therapeutic targets, mechanisms of resistance to therapy, tracking of evolutionary change and biomarker refinement in NEN.

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