206P - Characterisation of tumour-infiltrating lymphocytes (TILs) in liver metastases (LM) and primary tumour (PT) of microsatellite stable (MSS) colorectal cancers

Background

Tumour-specific TILs exert tumour control, but may be excluded or immunosuppressed by the tumour microenvironment, leading to ineffective tumour elimination. This study aims to characterise TILs in liver metastases (LM) and primary tumour (PT) of stage IV microsatellite-stable (MSS) colorectal cancers with liver metastases (CRLM), with the aim of overcoming poor response to immunotherapy.

Methods

Twelve patients with resectable CRLM were consented — five with metachronous LM; seven synchronous LM. Fresh tissues were obtained from PT, LM and adjacent normal tissues in six patients, with TIL/organoid cultures performed. Bulk TILs & CD39+CD103+ sorted CD8 TILs were expanded, and underwent FACS analyses and TCRsequencing. Fresh frozen samples from all patients underwent whole exome and RNA sequencing. Spatial transcriptomic profiling and single cell RNA sequencing (10X Genomics) were performed on selected patients.

Results

Flow cytometry analyses and T cell receptor (TCR) sequencing of TILs revealed differences in LM and PT. Post CD39+CD103+ sorting, LM TILs had higher proportions of CD39+LAG3+PD1+ triple-positive CD8 cells (p < 0.0001) and CD39+TIGIT+PD1+ cells (p< 0.05) compared to PT TILs, suggesting a potential tumour-specific population with high activation/exhaustion markers. Bulk LM TILs also had lower CD8 and higher proportion of CD4 cells compared to PT TILs. Organoid killing assays from patient 4108 demonstrated that LM TILs recognised and resulted in more killing of both LM and PT organoids than PT derived TILs. Spatial transcriptomic analyses showed immune exclusion of T cells in LM and distinct macrophage markers which may reveal potential immunosuppressive factors. WES/RNAseq revealed a similar neoantigen landscape between PT and LM, suggesting a possibility for a unifying therapeutic target. Single cell RNAseq results are still underway but preliminary bulk TCRseq from CD39+CD103+ CD8 T cells in PT and LM TILs showed overlap in a few TCR clones, suggesting shared TCR lineage.

Conclusions

Better understanding of the LM TIL and shared neoantigen landscape of CRLM supports its potential for TIL therapy and TCR discovery.

Legal entity responsible for the study

NMRC.

Funding

NMRC.

Disclosure

All authors have declared no conflicts of interest.