Background: Programmed death-1/programmed death ligand 1 (PD-[L]1) inhibitors are approved for use in a range of cancers. PD-L1 expression in the tumor microenvironment, assessed with an FDA-approved PD-L1 immunohistochemistry (IHC) diagnostic assay such as the Dako PD-L1 IHC 28-8 and 22C3 pharmDx or Ventana PD-L1 SP142 and SP263 assays, is associated with improved PD-(L)1 inhibitor treatment outcomes in some tumor types, including breast cancer (BC). In March 2019, the FDA approved atezolizumab + nab-paclitaxel for the treatment of patients with advanced triple-negative BC and immune cell (IC) PD-L1 expression ≥ 1% using the SP142 assay. Here, we investigated test utilization, test turnaround time (TAT), PD-L1 expression prevalence by assay and biopsy location, and analytical concordance between assays in real-world BC samples. Design: The study included samples from patients with BC that were tested for PD-L1 expression between Oct 2015 and Sep 2019 at NeoGenomics Laboratories, a US national reference laboratory. Patient characteristics from Symphony Healthcare Solutions were matched to PD-L1 test results using unique identifiers. Test volume and TAT were assessed for the 28-8, 22C3, SP142, and SP263 assays. PD-L1 expression was determined by trained pathologists using the 28-8, 22C3, or SP142 assays. Results for the 28-8 assay for the entire study period and for the 22C3 assay until Dec 2018 were reported as the percentage of tumor cells (% TC) with PD-L1 expression. From Jan 2019 onwards, 22C3 assay results were reported as a combined positive score (CPS). All SP142 assay results were reported as the percentage of ICs (% IC) with PD-L1 expression. Analytical concordance between assays was assessed in patients with matched samples (biopsies from the same site and collected on the same date). BioStat Solutions performed statistical analyses. Results: 2955 PD-L1 tests were performed on samples from 2508 patients with BC. The volume of PD-L1 tests on BC samples increased > 100-fold over the study period. Mean TAT was < 5 days for all 4 assays pooled. Table 1 shows PD-L1 expression prevalence in patients with a 28-8, 22C3, or SP142 test result. Median PD-L1 expression did not differ between primary tumors and metastatic sites. In matched samples, overall percentage agreement (OPA) between the 28-8 (TC ≥ 1%) and 22C3 (CPS ≥ 1) assays was 94%, and OPA between the 22C3 (CPS ≥ 1) and SP142 (IC ≥ 1%) assays was 64% (Table 2). Analytical concordance between the 28-8 and 22C3 assays for % TC scoring in matched samples from 27 patients was high (Kendall’s tau = 0.997 [95% CI, 0.883-1.000]). Conclusion: Mean PD-L1 test TAT for BC samples remained < 5 days across all tests despite a large increase in test volume over the study period. Prevalence of PD-L1 expression ≥ 1(%) was higher with the CPS and % IC algorithms than the % TC algorithm, although differences could be due to multiple confounding factors. Despite a small sample size, analytical concordance between the 28-8 and 22C3 assays in matched samples was high. These data provide real-world context for the PD-L1 testing landscape in BC. Table 1. Prevalence of PD-L1 expression in patients with BCPD-L1 expressiona28-8 and 22C3b22C3cSP142% TC, n (%)CPS, n (%)% IC, n (%)(N = 608)(N = 609)(N = 1080) Table 2. Agreement between assays on matched samples from patients with BCAgreement between 28-8 (TC ≥ 1%) and 22C3 (CPS ≥ 1) (N = 18)a28-8 as reference22C3 as referenceOPA (n/N)94 (17/18)PPA (n/N)100 (6/6)86 (6/7)NPA (n/N)92 (11/12)100 (11/11)Agreement between 22C3 (CPS ≥ 1) and SP142 (IC ≥ 1%) (N = 33)b22C3 as referenceSP142 as referenceOPA (n/N)64 (21/33)PPA (n/N)86 (12/14)55 (12/22)NPA (n/N)47 (9/19)82 (9/11)aData are presented for 28-8 and 22C3 tests on matched samples with 22C3 tests performed between Q1 2019 and Q4 2019; bData are presented for 22C3 and SP142 tests on matched samples with 22C3 tests performed between Q1 2019 and Q4 2019. N, total number of samples; n, number of samples with the same results with the 2 tests; NPA, negative percentage agreement; PPA, positive percentage agreement. Citation Format: Shreya Mitra, Emily A. Prince, James Pratt, James Novotny, Jr, Vladislav Chizhevsky, Josette William Ragheb, David Huron. Real-world PD-L1 test utilization and analytical concordance of the PD-L1 IHC 28-8 and 22C3 assays in patients with breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-18.