573P Multiparameter immunoprofiling reveals independent prognostic value of intraepithelial CD8 and intrastromal FoxP3 infiltrate in stage II/III colorectal cancer: Analysis of 3,409 cases from the SCOT and QUASAR2 trials

Tumour infiltrating CD8+ cytotoxic T cells confer favourable prognosis in colorectal cancer (CRC). The added prognostic value of other immune cells is unclear. We used multiparameter co-immunofluorescence to determine the density of CD8+, CD20+, FoxP3+ and CD68+ cells in the intraepithelial (IE) and intrastromal (IS) compartments in stage II/III CRCs from the SCOT and QUASAR2 trials. We determined marker prognostic and predictive value by Cox regression of SCOT cases, with validation in the QUASAR2 cohort. Our study included 2,340 SCOT and 1,069 QUASAR2 cases. Univariable analysis of recurrence-free interval (RFI) in SCOT revealed expected prognostic value of intraepithelial CD8 (CD8IE) (continuous variable: HR for cases at 75th vs 25th percentile (HR75vs25)= 0.73, 95% CI=0.68-0.79, P=2.5e-16) and, less expectedly FoxP3 within tumour stroma (FoxP3IS) (HR75vs25)= 0.71, 95% CI=0.64-0.78, P=1.5e-13), but not epithelium. CD8IE and FoxP3IS were independently prognostic in multivariable analysis, and a composite marker including both (CD8IE-Foxp3IS) was superior to either alone as a continuous variable (adjHR75vs25= 0.70; 95%CI=0.63–0.78; P=5.1e-11), and when categorised into three groups (Low/Int/High) using sample medians (Int vs High HR = 1.60, 95%CI=1.24–2.05, P=2.5e-04; Low vs High HR = 2.30, 95%CI=1.80–2.95; P=3.6e-11). These results were validated in the QUASAR2 trial. Pooled analysis of both studies revealed interactions between CD8IE-FoxP3IS and tumour sidedness (adjPINT=0.010), stromal proportion (adjPINT=0.018), and MMR status (adjPINT=0.079). CD8IE-FoxP3IS refined stage III risk groups, with clinical low risk (T1-3, N1), CD8IE-FoxP3IS low cases demonstrating similar 3-year recurrence-free probability to clinical high risk (T4 and/or N2), CD8IE-FoxP3IS high cases (79.2 vs 76.4%, adj log-rank P=0.16). Combined evaluation of CD8IE and FoxP3IS is superior to single markers and refines CRC risk stratification. FoxP3IS prognostic value is inconsistent with an immunosuppressive role; further investigation of these cells as an immunotherapy target is merited.