Investigating the immune landscape in gastric cancer
Document TypePhD thesis
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© 2019 Minyu Wang
This thesis investigates the relationship between the immunological microenvironment and clinical outcomes of patients diagnosed with gastric cancer (GC). I conducted a comprehensive study integrating immune cellular and molecular analyses of tumour tissues as well as paired peripheral bloods to investigate the role of T cells on clinical outcomes. Gene expression data from gastric tumours (n=100) and non-tumour gastric tissue (n=50) from a prospectively collected cohort (MAUGIC) was analysed using multiple bioinformatics tools to reveal that immune-related pathways and genes were enriched in gastric tumours. This enrichment emphasise T cells play a fundamental role in tumour biology and warrant detailed examination. The immune landscape of gastric cancer was based on a whole-slide multiplex immunohistochemistry (mIHC) platform which allowed both density and distance analysis of immunological components of the microenvironment. A novel algorithm measuring spatial distance relationships between cells, termed Intercellular Spatial Analysis Tool (ISAT) was developed. ISAT calculated the parameter, median intercellular nearest (MIN) distance, to reveal spatial characteristics relevant to dynamics of the tumour microenvironment. It was revealed that the EBV positive and microsatellite unstable (MSI) molecular subtypes showed a robust immune response. The difference in immune characteristics was not strong between intestinal and diffuse subtypes. The association of gastric cancer patients’ outcomes and the immune context in the tumours was further explored using mIHC in FFPE sections (n=56) and the transcriptome profiling data from paired tumours (n=40). It was discovered increased CD4+FOXP3+ T cell density in tumour correlated with prolonged survival. ISAT algorithm revealed CD4+FOXP3+ T cells clustered with CD8+ T cells rather than tumour cells. High density of CD4+FOXP3+ T cells and CD8+ T cells (High-High) predicted prolonged patient survival, and this was validated in an independent cohort (n=84). Gene expression profiling from paired tumour samples showed an interferon-gamma gene signature that was up-regulated in these High-High tumours and this gene signature was validated in two public gastric cancer datasets (n=876 total). Importantly, it was further revealed the High-High group also had prognostic benefit in genomically stable (GS) and chromosomal instability (CIN) molecular groups. No data to date showed these two groups were associated with activated host immunity. However, CIN and GS tumours represent the majority of gastric cancer patients (70% combined). The potential immune dysfunction mechanism/s in the High-High and Low-Low GS/CIN tumours on the systemic level was further explored by T-cell receptor sequencing and mass cytometry techniques using tumour tissue and matched peripheral blood. It was revealed that the High-High GS/CIN tumours were coupled with an increased interferon-gamma response, antigen presentation, dendritic cell differentiation and PDL1 up-regulation in the local tumours, as well as enrichment of Tbet+ CD4+ T cells and central memory CD4+ T cells circulating in the peripheral blood. In contrast, the Low-Low GS/CIN exhibited a high frequency of PDL1+ dendritic cells in the peripheral blood and low immune infiltrates in the tumour. In conclusion, using a combination of gene expression analysis, multiplex immunohistochemistry, T-cell receptor sequencing and mass cytometry, I have found a novel immunological clustering between CD8+ T cells and CD4+FOXP3+ T cells that identify patients with good prognosis and may serve as a novel biomarker to broaden targeting of immune checkpoint therapy beyond EBV/MSI GC, to include a significant number of GC patients with GS/CIN subtype. The findings from this study will significantly facilitate disease diagnosis and prognosis and aid in establishing precise immunotherapy treatment to individual gastric cancer patients, concurrently improving their overall survival outcome.
KeywordsGastric cancer; Immune response; Tumour microenvironment; CD8+ T cells; CD4+FOXP3+ T cells; Prognosis
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