cancers Article Discordance of PD-L1 Expression at the Protein and RNA Levels in Early Breast Cancer Ioannis Zerdes 1,2 , Vaia Karafousia 1 , Artur Mezheyeuski 3 , Maria Stogiannitsi 1 , Raoul Kuiper 4 , Pablo Moreno Ruiz 1 , George Rassidakis 1,5 , Jonas Bergh 1,2 , Thomas Hatschek 1,2 , Theodoros Foukakis 1,2 and Alexios Matikas 1,2, *   Citation: Zerdes, I.; Karafousia, V.; Mezheyeuski, A.; Stogiannitsi, M.; Kuiper, R.; Moreno Ruiz, P.; Rassidakis, G.; Bergh, J.; Hatschek, T.; Foukakis, T.; et al. Discordance of PD-L1 Expression at the Protein and RNA Levels in Early Breast Cancer. Cancers 2021, 13, 4655. https:// doi.org/10.3390/cancers13184655 Academic Editor: Carolien H. M. van Deurzen Received: 18 July 2021 Accepted: 13 September 2021 Published: 16 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Oncology-Pathology, Karolinska Institutet, 171 77 Stockholm, Sweden; ioannis.zerdes@ki.se (I.Z.); vkarafousia@gmail.com (V.K.); stogiam@hotmail.com (M.S.); pablo.moreno.ruiz@ki.se (P.M.R.); georgios.rassidakis@ki.se (G.R.); jonas.bergh@ki.se (J.B.); thomas.hatschek@ki.se (T.H.); theodoros.foukakis@ki.se (T.F.) 2 Breast Center, Theme Cancer, Karolinska University Hospital, 171 77 Stockholm, Sweden 3 Departmentof Immunology, Geneticsand Pathology, Uppsala University, P.O. Box 516, 751 20 Uppsala, Sweden; artur.mezheyeuski@igp.uu.se 4 Department of Laboratory Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden; raoul.kuiper@ki.se 5 Department of Pathology and Cytology, Karolinska University Hospital, 171 77 Stockholm, Sweden * Correspondence: alexios.matikas@ki.se; Tel.: +46-767-823-322 Simple Summary: Despite the increasing use of checkpoint inhibitors for early and metastatic breast cancer, Programmed Death Ligand 1 (PD-L1) remains the only validated albeit imperfect predictive biomarker. Significant discordance in PD-L1 protein expression depending on the antibody used has been demonstrated, while the weak correlation and discordant prognostic information between protein and gene expression underscore its biologic heterogeneity. In this study, we use material from two patient cohorts of early breast cancer and multiple methodologies (immunohistochemistry, RNA fluorescent in situ hybridization, immunofluorescence, bulk gene expression, and multiplex fluorescent immunohistochemistry) to demonstrate the significant discordance in PD-L1 expression among various methods and between different areas of the same tumor, which hints toward the presence of spatial, intratumoral and biological heterogeneity. Abstract: We aimed to assess if the discrepant prognostic information between Programmed Death Ligand 1 (PD-L1) protein versus mRNA expression in early breast cancer (BC) could be attributed to heterogeneity in its expression. PD-L1 protein and mRNA expression in BC tissue microarrays from two clinical patient cohorts were evaluated (105 patients; cohort 1: untreated; cohort 2: neoadjuvant chemotherapy-treated). Immunohistochemistry (IHC) with SP142, SP263 was performed. PD-L1 mRNA was evaluated using bulk gene expression and RNA-FISH RNAscope ® , the latter scored in a semi-quantitative manner and combined with immunofluorescence (IF) staining for the simultaneous detection of PD-L1 protein expression. PD-L1 expression was assessed in cores as a whole and in two regions of interest (ROI) from the same core. The cell origin of PD-L1 expression was evaluated using multiplex fluorescent IHC. IHC PD-L1 expression between SP142 and SP263 was concordant in 86.7% of cores (p < 0.001). PD-L1 IF/IHC was weakly correlated with spatial mRNA expression (concordance 54.6–71.2%). PD-L1 was mostly expressed by lymphocytes intra-tumorally, while its stromal expression was mostly observed in macrophages. Our results demonstrate only moderate concordance between the various methods of assessing PD-L1 expression at the protein and mRNA levels, which may be attributed to both analytical performance and spatial heterogeneity. Keywords: breast cancer; heterogeneity; in situ hybridization; multiplex immunohistochemistry; PD-L1 Cancers 2021, 13, 4655. https://doi.org/10.3390/cancers13184655 https://www.mdpi.com/journal/cancers