Digital spatial profiling: Mapping tumor responses to radiotherapy in rectal cancer.

3593 Background: Variation in response to radiotherapy for the treatment of rectal cancer is likely due to heterogeneity in the tumour microenvironment. However, to date, no reliable predictive biomarkers of response are in clinical use and the mechanisms underlying response are unknown. Tertiary lymphoid structures (TLS), which are ectopic lymphoid aggregates found in the tumour microenvironment, have been linked to response to immunotherapy, but little is known about their role in radiotherapy. Here, we aimed to explore the potential of lymphocytes and tertiary lymphoid structures as predictive biomarkers of response to radiotherapy and profile the tumour immune microenvironment in the context of response to radiotherapy. Methods: For this study, we accessed pre-treatment biopsies from 20 rectal cancer patients with known pathological response to long-course chemoradiotherapy (LCCRT). We selected regions of interest based on immunohistological identification of tumour and lymphocytic infiltrate in formalin-fixed paraffin-embedded tissue. We performed targeted proteomic profiling of 87 immuno-oncology proteins using the Nanostring GeoMx Digital Spatial Profiler to quantify protein expression with spatial resolution within regions of interest, including TLSs, in the tumour microenvironment. Results: Unsupervised clustering based on normalised protein expression showed a clear separation between the complete responders to LCCRT and all other tumours, and this separation is driven by differences in T cells within TLSs (CD3+). Differentially expressed proteins within CD3+ aggregates include depletion of the natural killer cell marker, CD56 and increased expression of the apoptosis marker, cleaved caspase 9. The distribution of TLS-tumour distance was also significantly different between response groups. Conclusions: The study highlights the role of TLSs in modulating the immunogenic landscape of the tumour microenvironment in rectal cancer, likely influencing the response to radiotherapy. Spatially resolved proteomic analyses identifies potential biomarkers for radiotherapy response and underscores the importance of profiling tumour-immune microenvironment complexity when stratifying patients for therapy.