217P Heterogeneity of PD-L1 expression at the protein and mRNA levels in early breast cancer

Abstract

We have previously shown that PD-L1 confers discrepant prognostic information depending on level of expression, protein versus mRNA. This discrepancy may be due to heterogeneity in its expression, which we aimed to assess. Breast cancer samples from an adjuvant patient cohort (cohort 1; surgical specimen, pre-adjuvant) and from a prospective single-arm study of neoadjuvant chemotherapy (cohort 2; multiple timepoints during chemotherapy) were obtained and Tissue Microarrays (TMA) were constructed. Protein and mRNA PD-L1 expression were assessed in two different areas of each sample, each containing at least 50 cells. Immunofluorescence (IF) staining with anti-PD-L1 (clone SP142, Abcam) antibody was performed. A core was considered as protein PD-L1 positive when at least one cell with membranous immunostaining was detected in at least one TMA core, regardless of cell of origin (tumor or immune cell). PD-L1 mRNA was simultaneously detected using the in situ hybridization method RNAscope® (Multiplex Fluorescent v2 Assay, Advanced Cell Diagnostics Inc) and quantified in a semi-quantitative manner. A total of 90 patient samples were analyzed (cohort 1: 33, cohort 2: 57). At the protein level, 53% of areas in cohort 1 and 42.9% in cohort 2 had at least one PD-L1 positive cell, with a mean positivity rate of 6.11% and 3.99% cells per area, respectively. There was no evidence of heterogeneity in the protein expression between two areas of the same sample (cohort 1: Wilcoxon signed rank p=0.171; cohort 2: p=0.447). At the mRNA level, 69.7% of areas scored in cohort 1 and 81.3% in cohort 2 had none or very low staining (ACD score 0). PD-L1 mRNA expression scores between the two areas of each sample were statistically significantly but modestly correlated to each other both in cohort 1 (Spearman’s rho=0.40, p=0.021) and cohort 2 (Spearman’s rho=0.40, p=0.002). PD-L1 protein expression was strongly and statistically significantly correlated with mRNA expression (cohort 1: Spearman’s rho=0.805, p<0.001; cohort 2: Spearman’s rho=0.731, p<0.001). Our results showed high concordance between mRNA-based and protein-based assessment of PD-L1. No evidence was found in this study for neither spatial nor biological heterogeneity of PD-L1 expression.