Immune profiling of oral pre-malignant lesions (OPLs): An Erlotinib Prevention of Oral Cancer (EPOC) study biobank analysis.

Abstract

1545 Background: We previously demonstrated that high-risk loss of heterozygosity (LOH) profiles (i.e., 3p14/9p21 LOH) and EGFR gene copy number gain (CNG) in OPLs were associated with inferior oral cancer-free survival (OCFS) in patients enrolled in the randomized EPOC trial. Herein, we performed comprehensive immune profiling of OPLs and correlated the findings with molecular features and outcomes, using the prospectively collected and clinically annotated EPOC biobank. Methods: We evaluated OPL specimens by multiplex immunofluorescence using the Opal 7-color fIHC Kit and the Vectra multispectral microscope / inForm Cell Analysis software. Markers included AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), CD3, CD8, and CD68. Wilcoxon rank-sum and Fisher’s exact tests were used to assess the associations between binary markers and continuous and categorical variables, respectively. Cox model was used to investigate associations of markers with OCFS. Results: The cohort included 188 OPL patients with hyperkeratosis/hyperplasia (18%), mild/moderate (44%), or severe dysplasia (5%); 65% had high-risk LOH profiles. The 5-year OCFS was 72.3% (median follow-up of 50 months). PD-L1 expression in > 1% of epithelial cells occurred in 28% of OPLs. Intraepithelial CD3+, CD3+/CD8+, CD68+, and CD68+/PD-L1+ cells were detected in 100%, 88%, 88%, and 54% of the samples, respectively. OPLs with high-risk LOH profiles had increased epithelial PD-L1 expression (P = 0.007), intraepithelial CD68+/PD-L1+ cells (P = 0.002), and a trend towards more CD3+/CD8+ cells in the stroma (P = 0.06) but not in the epithelium (P = 0.97), compared with low-risk LOH OPLs. Increased epithelial PD-L1 expression was associated with inferior OCFS on univariate (P = 0.023), and multivariate analysis including LOH status and EGFR CNG as co-variates (P = 0.018). Conclusions: High-risk OPLs defined by LOH profiles had increased PD-L1 expression in epithelial cells and intraepithelial macrophages, as well as stromal CD3+/CD8+ immune infiltration. Higher PD-L1 expression was associated with increased oral cancer risk. The findings may support evaluation of (PD-1-targeted) immunoprevention strategies in high-risk OPLs.