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

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

Smartphone and medical related App use among medical students and junior doctors in the United Kingdom (UK): a regional survey

Background: Smartphone usage has spread to many settings including that of healthcare with numerous potential and realised benefits. The ability to download custom-built software applications (apps) has created a new wealth of clinical resources available to healthcare staff, providing evidence-based decisional tools to reduce medical errors. Previous literature has examined how smartphones can be utilised by both medical student and doctor populations, to enhance educational and workplace activities, with the potential to improve overall patient care. However, this literature has not examined smartphone acceptance and patterns of medical app usage within the student and junior doctor populations. Methods: An online survey of medical student and foundation level junior doctor cohorts was undertaken within one United Kingdom healthcare region. Participants were asked whether they owned a Smartphone and if they used apps on their Smartphones to support their education and practice activities. Frequency of use and type of app used was also investigated. Open response questions explored participants’ views on apps that were desired or recommended and the characteristics of apps that were useful. Results: 257 medical students and 131 junior doctors responded, equating to a response rate of 15.0% and 21.8% respectively. 79.0% (n=203/257) of medical students and 74.8% (n=98/131) of junior doctors owned a smartphone, with 56.6% (n=115/203) of students and 68.4% (n=67/98) of doctors owning an iPhone. The majority of students and doctors owned 1–5 medical related applications, with very few owning more than 10, and iPhone owners significantly more likely to own apps (Chi sq, p<0.001). Both populations showed similar trends of app usage of several times a day. Over 24hours apps were used for between 1–30 minutes for students and 1–20 minutes for doctors, students used disease diagnosis/management and drug reference apps, with doctors favouring clinical score/calculator apps. Conclusions: This study found a high level of smartphone ownership and usage among medical students and junior doctors. Both groups endorse the development of more apps to support their education and clinical practice