Characterisation of circulating tumour cells in metastatic prostate cancer

Abstract

In the UK, prostate cancer is the most prevalent male cancer and approximately 40% of men will have metastatic disease at diagnosis. The treatment pathway in metastatic prostate cancer is offered often without any histological or genetic knowledge of the tumour and there is currently no reliable biomarker to monitor response to treatment. Over the past decade, there has been increasing interest in circulating tumour cells (CTCs). These cells, which have broken away from the tumour of origin and can be captured via a simple blood test, can be quantified, sequenced or examined for antigen expression. Stem cell marker expression, specifically Oct4, SOX2 and Nanog, has been found to correlate with aggressive disease when looking at solid prostate tissue. Consequently, exploring the expression of these markers in circulating tumour cells could enable the development of a new biomarker in prostate cancer. This study had three aims. The first was to optimise an assay using flow cytometry to enable detection of the stem cell markers Oct4, SOX2 and Nanog, alongside epithelial and mesenchymal markers in CTCs from patients with metastatic prostate cancer. The second aim was to explore the prognostic role of these markers and the final aim was to culture CTCs from patients to enable downstream utilisation. Blood was obtained from seventy-eight patients with different stages of prostate cancer and processed using two flow-cytometry based methods; one on the Imagestream, a combined flow cytometer and high-resolution microscope, and the second using conventional multi-channel FACS. Enumeration of total number of CTCs in addition to the individual marker positive cells was correlated with existing clinical data (PSA and Alkaline Phosphatase level, in addition to survival). Cells from six patients were successfully maintained in culture for up to a year. Attempts to prove the genotype of these cells included real time qPCR, SNP Array and whole exome sequencing experiments. Cells from one patient were implanted into five NSG mice and experiments to look at both chemokine expression and the Young’s modulus of the cells were also performed. No correlation was found between either CTC count or antigen expression and the clinical outcome of the patients. Unfortunately, due to a lack of good quality DNA the sequencing ii experiments didn’t yield any data and despite one of the mice developing a hind leg paralysis, there was no histological evidence of an engrafted tumour. The chemokine receptor and Young’s modulus experiments showed promising early results and form part of some ongoing collaborative projects. The first aim of developing an assay to detect prostate cancer CTCs was achieved, and survival data at a later time point is going to be collected to ascertain whether the markers explored in this study have a prognostic role. Whilst cells were successfully cultured, the genetic signature of these is still unclear and future plans to continue this work are currently in progress.