Background <p>Curative-intent surgery is the standard treatment for node-negative, early-stage rectal cancer. Although distant recurrence is uncommon in this setting, it carries a markedly poor prognosis. There is limited understanding of tumour biology linked to this aggressive cancer and no biomarkers to aid clinical practice. The aim of this pilot study was to demonstrate feasibility to identify putative prognostic biomarkers of distant metastasis following curative-intent surgery for early rectal cancer.</p> Methods <p>Treatment-naïve patients who developed distant metastases after total mesorectal excision for Stage I/IIA rectal cancer were identified from our centre over a 7-year period. A cohort matched for clinicopathological features without subsequent metastasis was used as control. Formalin-fixed paraffin embedded primary tumour was laser microdissected and proteins extracted for mass-spectrometry based proteomics. Separately, intratumoural CD3⁺, CD3⁺CD8⁺ and CD3⁺CD8⁻ T cells densities were measured using immunohistochemistry.</p> Results <p>In our cohort, the frequency of distant metastasis was 8.4%. 5,473 proteins were quantified in all samples, with 69 proteins differentially expressed between primary tumours with or without subsequent distant metastasis. TGF-β signaling, epithelial-mesenchymal transition, and coagulation pathways were enriched in primary tumours that developed distant metastases. Total T cells and CD8^– T cell densities were lower in both the epithelium and stroma of patients with distant metastasis. CD8⁺ T cell density was also lower in the tumour epithelial regions of patients who developed distant metastases.</p> Conclusions <p>We demonstrated feasibility to detect putative protein and T cell infiltrate biomarkers of distant metastasis in early-stage rectal cancer. A multi-centre validation study is now required to identify higher risk patients where adjuvant chemotherapy may improve outcomes.</p>