CONTEXT.—:Regulatory approval of pembrolizumab for treatment of gastric and gastroesophageal junction (G/GEJ) adenocarcinoma required a reproducible scoring method for use of programmed death ligand-1 (PD-L1) protein expression as a companion diagnostic to identify likely responders to therapy.OBJECTIVE.—:To develop an immunohistochemical scoring algorithm that includes PD-L1 expression for tumor and immune cells, that is, the combined positive score.DESIGN.—:Four previously treated tumor types in the KEYNOTE-012 and KEYNOTE-028 studies were analyzed descriptively with a version of the PD-L1 immunohistochemical 22C3 pharmDx assay labeled for investigational use only to determine the relative importance of PD-L1 expression in tumor versus immune cells as a biomarker for pembrolizumab response. A combined positive score was developed as a novel scoring method and was compared with the tumor proportion score in cohort 1 from the KEYNOTE-059 study (G/GEJ cancer). External reproducibility was assessed.RESULTS.—:Per combined positive score cutoff of 1 or more, the prevalence of PD-L1 expression in patients with G/GEJ cancer was 57.6% (148 of 257 patients), with reasonable enrichment of responses (odds ratio, 2.8). Per tumor proportion score cutoff of 1% or more, prevalence was 12.5% (32 of 257 patients), with minimal enrichment (odds ratio, 1.4). External reproducibility assessments demonstrated interpathologist overall agreement of 96.6% (591 of 612; 95% CI, 94.0%-98.7%) and intrapathologist overall agreement of 97.2% (595 of 612; 95% CI, 95.3%-98.9%).CONCLUSIONS.—:Combined positive score is a robust, reproducible PD-L1 scoring method that predicts response to pembrolizumab in patients with G/GEJ cancer. This novel scoring method supported US Food and Drug Administration approval of pembrolizumab as third-line therapy for G/GEJ cancer and has facilitated investigation in other indications.

e14585 Background: PD-L1 IHC 28-8 pharmDx is a qualitative assay developed by Agilent Technologies for the Autostainer Link 48 platform and is based on EnVision FLEX visualization technology and monoclonal rabbit anti-PD-L1, clone 28-8 antibody. The assay has been co-developed with the immunotherapeutic agent nivolumab; initially as an aid in assessing PD-L1 expression in non-squamous non-small cell lung cancer (NSCLC) and melanoma patients. Here we describe the efforts to validate this assay for urothelial carcinoma (UC). Methods: IHC staining was performed on Autostainer Link 48 platform using an automated staining protocol stated per the assay’s instruction for use (IFU). Specimens were coverslipped and interpreted for % PD-L1 positive tumor cells. The assay was analytically validated on commercially acquired formalin-fixed, paraffin-embedded (FFPE) human UC invasive tumor specimens at ≥1% and ≥5% PD-L1 positive tumor cells expression levels. Results: A wide range of % PD-L1 positive tumor cells at all staining intensity levels have been detected. Assay in-house precision was validated for inter-operator, inter-instrument, inter-day, inter-run and intra-run as well as inter-observer and intra-observer agreement. Robustness studies evaluated the assay under multiple conditions for target retrieval pH, temperature and incubation time, slide type as well as tissue section thickness. Assay reproducibility was evaluated at three external sites by testing samples for intra-site/inter-day and inter-site agreement measures. Specimens were also evaluated by an observer at each site, with three reads for each observer to assess intra-observer and inter-observer agreement. All validation studies demonstrated agreement estimates above 85% with values for lower bound 95% confidence intervals calculated above 84%. Conclusions: Results of all conducted studies show high robustness and reproducibility of the assay on UC.

e14588 Background: The PD-L1 IHC 28-8 pharmDx assay has been developed for use in detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-squamous NSCLC and melanoma tissues for associated treatment effect with nivolumab. Validation of the assay for measuring PD-L1 expression level in human squamous cell carcinoma of the head and neck (SCCHN) FFPE sections has been conducted and results are described. Methods: Visualization of the PD-L1 IHC 28-8 pharmDx assay is based on the EnVision FLEX IHC technology. As described in the Instructions For Use, antigen retrieval was performed using the Dako PT Link, and the automated staining was conducted with Autostainer Link 48. The assay performance was validated on commercially procured FFPE SCCHN specimens. Results: Assessment of assay sensitivity to PD-L1 expression was measured on 236 unique specimens originating from squamous cell carcinoma of the tongue, tonsil, nasopharynx, oropharynx, hypopharynx, and larynx. The study demonstrated PD-L1 positive staining across a range of 0-95% positive tumor cells and 0 to 3+ staining intensity. Among these tested specimens, 44% of specimens had PD-L1 expression levels ≥1%. Assay robustness was validated by assessing critical parameters such as target retrieval solution pH, target retrieval solution temperature, target retrieval time, and cut section thickness. Assay reproducibility was evaluated in a study at three external sites. Reproducibility of intra-site, inter-day, and inter-site agreement was assessed in addition to inter- and intra-observer agreement. Assay robustness and reproducibility demonstrated agreement estimates above 85% with values for lower bound 95% confidence intervals calculated above 85%. Conclusions: These studies demonstrate that the PD-L1 IHC 28-8 pharmDx assay is reproducible and robust when used for automated detection of PD-L1 protein in FFPE human SCCHN specimens using Autostainer Link 48.