A DNA methylation driven study into immunotherapy response in oesophageal adenocarcinoma patients and a spatiotemporal investigation of foetal oesophageal development

Oesophageal adenocarcinoma (OAC) is a cancer of the oesophagus occurring around the gastro-oesophageal junction and is a deadly disease with a rapidly increasing prevalence in the Western world. Many cases are already advanced by the time they are detected and, for such patients, the current standard treatment is surgery with chemotherapy or chemoradiotherapy. The outcomes with the standard first line treatments are poor, with the overall 5 year survival rate around 20 percent. So, the LUD2015-005 clinical trial – a phase 1/2 study of checkpoint blockade anti-PD-L1 (or CTLA4) in combination with chemo(radio)therapy – considered the potential benefits of immunotherapy through a collaborative multi-omics approach. In this thesis, a particular focus is placed on DNA methylation, along with connections to gene expression. Using a novel whole genome DNA methylation sequencing method called TAPS (TET-assisted pyridine borane sequencing), patient samples were examined across various treatment time points. Notably, the patients with hypomethylated tumours prior to treatment seemed to have better clinical benefit. Furthermore, this work highlighted genes for further investigation as potential predictive markers of response or as important participants in the mechanism of response. The potential links between DNA methylation and RNA expression were also examined. Because normal oesophageal development and the process of tumourigenesis, from normal tissue through Barrett’s oesophagus to the eventual tumour, show tissue metaplasia occurring in opposite directions, it was suspected that similar processes may be involved. Because foetal oesophageal development is an understudied topic with outdated and dissonant data, a spatiotemporal approach was employed to better understand the changes involved. Extensive H&E and immunohistochemistry staining showed that, contrary to previous belief, the metaplasia from columnar to squamous epithelium occurs at similar rates regardless of the longitudinal position along the foetal oesophagus. Furthermore, no abrupt borders between epithelial types were observed, and the transition seemed to occur transiently through various stages of intermediate identity rather than an acute intrusion of squamous cells. A spatiotemporal RNAseq experiment, as well as analysis of a publicly available scRNAseq dataset, echoed and added further support to these results. Overall, this thesis has characterized important features in DNA methylation profiles and highlighted key genes that may be relevant in understanding immunotherapy response. In addition, it has provided the framework for investigating whether there are patterns implicated in oesophageal development that are aberrantly regulated in disease. Ultimately, it is hoped that this work can help tackle the unmet clinical need to identify the cause of OAC and give guidance to help enable stratification of high risk patients for surveillance and preventative treatment.