We then validated our results on a commercially available, genetically defined PD-L1 engineered cell collection array with a range of controlled protein expressing cell lines. with a range of controlled protein expressing cell lines. Protein levels were measured by both quantitative immunofluorescence and quantitative chromogenic assessment. Results Concordance between 4 antibodies showed regression (R2 ideals) between 0.42-0.91 for tumor cells cores and 0.83-0.97 for cells collection cores by QIF in the PD-L1 index cells microarray. All six antibodies showed high levels of concordance (R2 ranging from 0.76 to 0.99) when using chromogenic staining in isogenic cell lines. Conclusions and Relevance Since the antibodies are highly concordant, these results suggest that assays based on the use of these antibodies could yield concordant results. They further suggest that previously explained variations in PD-L1 manifestation in cells is independent of the antibody utilized and likely due to tumor heterogeneity, assay/platform-specific variables or other factors. To identify responders and lead treatment, assessment of PD-L1 expression by immunohistochemistry (IHC) has been used as a predictive diagnostic test in trials of PD-1 axis inhibitors nivolumab, atezolizumab, durvalumab and pembrolizumab in NSCLC (1-4). The PD-L1 IHC 22C3 PharmDx kit (Dako North America) was recently approved by the FDA as a companion diagnostic for pembrolizumab in NSCLC while PD-L1 28-8 PharmDx kit (Dako North America) was approved as a complementary diagnostic for nivolumab. The definition of PD-L1 positive R428 lacks standardization, and prediction of response by IHC is additionally limited by the subjective nature of the technique. Variable cutoffs for defining positive cases across trials have been utilized, measuring either tumor and/or immune cells in the stroma (4-6). While some PD-L1 antibodies have been rigorously validated in the published literature, including 28-8 and E1L3N, others are R428 less documented and specific epitope sequences remain proprietary (7, 8). Significant differences in case classification has been observed with two validated PD-L1 antibodies; concluding that discordance seen was either a function of tissue heterogeneity or variability between antibodies (9). To examine the effect of epitope targeting or potential non-specific binding, we developed a tissue microarray with a range of positive and negative specimens including tumor, normal tissue and cell lines (eFigure 1). We analyzed five PD-L1 monoclonal antibodies (intracellular and extracellular domain name specific) to determine the concordance between antibodies. Retrospectively collected formalin R428 fixed paraffin embedded tissue blocks from 30 patients were obtained from Yale Pathology Archives with a range of expression of PD-L1 as assessed in previous studies. All cases obtained had signed consent or waiver of consent for tissue use under approved Yale Human Investigation Committee protocol #9505008219. Tissue and cell collection blocks, processed identically, were prepared in a tissue microarray format made up of 0.6 mm representative FFPE cores in two-fold redundancy Horizon Discovery’s gene editing technology was used to develop a Rabbit Polyclonal to GJA3 genetically defined 15 spot cell collection microarray (CLMA) PD-L1 IHC Reference standard with a range of controlled protein expression levels (negative, low, medium and high protein expressing cell lines). Individual cell lines in the 15 spot CLMA were extensively characterized and verified using molecular assays, IHC and quantitative digital pathology. PD-L1 expression was evaluated by chromogenic immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) using six monoclonal antibodies raised against PD-L1 as summarized in Table 1. Antibodies were titrated at a range of concentrations and optimal assay concentration was R428 decided using an algorithm that utilizes both transmission to noise ratio and dynamic range. QIF was performed successfully for four of the six antibodies using AQUA as previously explained(9). Chromogenic IHC was quantified using the Aperio Positive Pixel Count based on the intensity of membrane staining of respective PD-L1 antibodies using a altered HER2 algorithm (10). A detailed description of these methods and immunostaining examples are included in supplemental materials. (eFigure 2 and 3). Table 1 Antibody Characteristics and concentrations tested to determine optimal concentration for QIF thead th valign=”top” align=”center” rowspan=”1″ colspan=”1″ Antibody /th th valign=”top” align=”center” rowspan=”1″ colspan=”1″ PD-L1 /th th valign=”top” align=”center” rowspan=”1″ colspan=”1″ PD-L1 /th th valign=”top” align=”center” rowspan=”1″ colspan=”1″ PD-L1 /th th valign=”top” align=”center” rowspan=”1″ colspan=”1″ PD-L1 /th th valign=”top” align=”center” rowspan=”1″ colspan=”1″ PD-L1 /th th valign=”top” align=”center” rowspan=”1″ colspan=”1″ PD-L1 /th /thead CloneE1L3NSP142405.9A11SP26328-822C3SourceCell Signaling Technology Inc.Spring Bioscience Corp.Cell Signaling Technology Inc.Ventana Medical Systems Inc.Abcam Plc.DakoIsotype and Host SpeciesRabbit IgGRabbit IgGMouseRabbit IgGRabbit IgGMouseBinding SiteIntracellularIntracellularIntracellularIntracellularExtracellularExtracellularNeat Antibody Concentration1010 g/ml77 g/ml100 ug/ml1.61 g/ml967 g/mlNPOptimal AntibodyConcentration1 g/ml0.1 g/ml1 g/mlPre-diluteUNP Open in a separate window Correlation between antibodies was measured by linear regression. Tumor cores showed lower concordance than cell lines.