Utility of a liquid biopsy to identify intra-tumoural molecular heterogeneity in head and neck squamous cell carcinoma: the role of circulating tumour cells

Payne, Karl Frederick Braekkan (2022). Utility of a liquid biopsy to identify intra-tumoural molecular heterogeneity in head and neck squamous cell carcinoma: the role of circulating tumour cells. University of Birmingham. Ph.D.

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BACKGROUND: Circulating tumour cells (CTCs) in the blood of cancer patients are a potentially rich source of biomarkers to guide cancer therapy, particularly when tumours cannot be sampled directly. The optimal method of CTC enrichment/isolation and subsequent downstream characterisation remains unclear. Current marker dependent platforms are biased towards specific CTC sub-groups, and CTC characterisation focuses on genomic characterisation, being unable to detect post-transcriptional modification.

AIMS: The aims of this project were: i) develop blood sampling methods able to preserve key CTC parameters with minimal sampling handling, ii) optimise a marker-independent CTC enrichment technique, the Parsortix microfluidic platform, for use in head and neck squamous cell carcinoma (HNSCC), iii) investigate methods for direct proteomic characterisation of CTCs to detect relevant post translational modifications. Collectively these aims would streamline sample collection thus simplifying and enhancing national/international multi-centre CTC research studies and trials while allowing deeper characterisation of CTC biology.

METHODS: A mock CTC model using the FaDu and SCC047 HNSCC cell lines spiked into healthy donor blood was developed and used to optimise the Parsortix platform and downstream immunofluorescence microscopy and flow cytometry characterisation. Subsequently, a mass cytometry antibody panel of 44 markers (including epithelial/EMT, proliferative, stemness, immune and phosphorylated signalling proteins) was optimised for staining quality on Transfix fixed cells, following Parsortix enrichment. Pre-treatment blood samples from HNSCC patients were prospectively recruited through the Accelerated2 sample collection platform.

RESULTS: In cell line spiking experiments, non-fixative EDTA blood collection tubes (BCTs) enriched with Parsortix demonstrated a mean capture rate of 53.5%, across a range of 9-129 cells/ml spiked concentrations (n=13). Transfix fixation BCTs demonstrated significantly improved capture rates of spiked cells at 0hr, 24hr and 72hr timepoints, when compared to EDTA BCTs. Parsortix enrichment significantly altered the gene expression of unfixed cells, causing downregulation of genes associated with RNA and ribosomal/protein processing genes and upregulation of genes associated with oxidative stress and cell injury/apoptosis. Using immunofluorescence microscopy, CTCs were identified from HNSCC patient samples (enriched with Parsortix) with epithelial (EpCAM expression) and epithelial-mesenchymal transition (EMT, EpCAM and N-cadherin expression) characteristics. In a cohort of 20 patients, flow cytometry characterisation of the above markers was able to quantify and characterise CTCs. Sixty-five percent (13/20) demonstrated CTCs, at a mean count of 4 CTCs/ml (range 2-11.2 CTCs/ml). The presence of CTCs correlated with advanced stage of disease (p=0.0121), but not T or N stage. Neither the presence of epithelial nor EMT CTC sub-groups correlated with progression-free or overall survival. A mesenchymal gene expression profile in patient matched primary tumour tissue did not positively correlate with CTC EMT expression (p=0.347). Mass cytometry analysis identified CTCs in 11 of 12 patient blood samples, with considerable CTC heterogeneity. Novel CTC sub-groups, common between patient samples, were defined based upon target marker expression patterns - for example EMT CTCs expressing proliferative markers and decreased immune-checkpoint markers. Mass cytometry outperformed bulk quantitative gene expression profiling of Parsortix enriched samples from the same cohort – both to identify the presence of CTCs and phenotypic sub-groups. A mesenchymal gene expression profile in primary tumour tissue was significantly associated with an increased proportion of proliferative and immune-checkpoint high CTCs.

DISCUSSION: We demonstrate for the first time that the combination of Parsortix microfluidic enrichment and mass cytometry can be successfully used to provide multiparameter single-CTC characterisation of extracellular, intracellular and activated signalling proteins – a depth of information beyond that available from bulk CTC gene expression profiling. Even in a small cohort pilot study, we highlight an interesting trend that primary tumours with mesenchymal gene expression profiles exhibit increased levels of proliferative and immune-checkpoint high CTCs. The heterogeneity of CTC EMT and phenotypic subgroups and their relationship to immune-checkpoint markers is notable – providing both an avenue for therapeutic targeting, but also a means of therapeutic escape in ‘immune low’ CTCs. We lay a platform for future proteomic based single-CTC studies in larger cohorts.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Medical & Dental Sciences
School or Department: Institute of Cancer and Genomic Sciences
Funders: Cancer Research UK, Other
Other Funders: Birmingham Health Partners, British Association of Oral and Maxillofacial Surgeons, British Association of Head and Neck Oncologists, Royal College of Surgeons of England
Subjects: R Medicine > RC Internal medicine
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
URI: http://etheses.bham.ac.uk/id/eprint/12677


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