The Blueprint PD-L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD-L1 expression for three assays, interchanging assays and cutoffs would lead to "misclassification" of PD-L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy-related PD-L1 cutoffs.

BackgroundA high-quality programmed cell-death ligand 1 (PD-L1) diagnostic assay may help predict which patients are more likely to respond to anti-programmed cell death-1 (PD-1)/PD-L1 antibody-based cancer therapy. Here we describe a PD-L1 immunohistochemical (IHC) staining protocol developed by Ventana Medical Systems Inc. and key analytical parameters of its use in formalin-fixed, paraffin-embedded (FFPE) samples of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC).MethodsAn anti-human PD-L1 rabbit monoclonal antibody (SP263) was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform. The VENTANA PD-L1 (SP263) Assay was validated for use with FFPE NSCLC and HNSCC tissue samples in a series of studies addressing sensitivity, specificity, robustness, and precision. Samples from a subset of 181 patients from a Phase 1/2 study of durvalumab (NCT01693562) were analyzed to determine the optimal PD-L1 staining cut-off for enriching the probability of responses to treatment. The scoring algorithm was defined using statistical analysis of clinical response data from this clinical trial and PD-L1 staining parameters in HNSCC and NSCLC tissue. Inter-reader agreement was established by three pathologists who evaluated 81 NSCLC and 100 HNSCC samples across the range of PD-L1 expression levels.ResultsThe VENTANA PD-L1 (SP263) Assay met all pre-defined acceptance criteria. For both cancer types, a cut-off of 25 % of tumor cells with PD-L1 membrane staining of any intensity best discriminated responders from nonresponders. Samples with staining above this value were deemed to have high PD-L1 expression, and those with staining below it were deemed to have low or no PD-L1 expression. Inter-reader agreement on PD-L1 status was 97 and 92 % for NSCLC and HNSCC, respectively.ConclusionsThese results highlight the robustness and reproducibility of the VENTANA PD-L1 (SP263) Assay and support its suitability for use in the evaluation of NSCLC and HNSCC FFPE tumor samples using the devised ≥25 % tumor cell staining cut-off in a clinical setting. The clinical utility of the PD-L1 diagnostic assay as a predictive biomarker will be further validated in ongoing durvalumab studies.Trial registrationClinicalTrials.gov: NCT01693562

BackgroundProgrammed Death Ligand 1 (PD-L1) is an immune modulating protein expressed on the surface of various inflammatory cells, including T Cells, B Cells, dendritic cells, and macrophages. PD-L1 represents an important diagnostic target; expression of PD-L1 on the surface of tumor cells, or within tumor-associated immune cells, is an important predictor of likely response to targeted therapies. In this study, we describe the optimization of immunohistochemistry (IHC) assays using two PD-L1 antibodies (SP263 and E1L3N) and compare the performance of the optimized assays.MethodsFully automated immunohistochemical assays were optimized for the VENTANA PD-L1 (SP263) Rabbit Monoclonal Antibody and the PD-L1 (E1L3N®) XP® Rabbit mAb using instruments and detection chemistries from Ventana Medical Systems, Inc. (“SP263 assay” and “E1L3N assay,” respectively). Tissue microarrays (TMAs) containing formalin fixed paraffin embedded (FFPE) non-small cell lung cancer (NSCLC) cases were used for the optimization and comparison staining. H scores were used for membrane scoring whereas percent positivity was used for tumor-associated immune cell scoring.ResultsOne-hundred NSCLC TMA case cores each stained with the SP263 and E1L3N assays were evaluated by two pathologists in a blinded study. Analysis of these specimens showed that the SP263 assay was more sensitive and had a wider dynamic range than the E1L3N assay. For sensitivity, many cases were found to be negative for membrane staining with the E1L3N assay, but had measurable staining with the SP263 assay. Dynamic range was demonstrated by the SP263 assay having well-distributed H scores while the E1L3N assay had a significantly higher proportion of cases clustered in the lowest H score bins. For tumor-associated immune cell staining, the two assays identified similar amounts of cells staining in each case, but the SP263 assay gave overall darker staining.ConclusionSince PD-L1 status is important for targeted therapies, having a specific and accurate diagnostic test is crucial for identifying patients who could benefit from these treatments. Due to its staining intensity, scoring range, and pathologist preference, the SP263 IHC assay has been deemed superior to the E1L3N IHC assay. Future clinical utility remains to be determined.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-016-0494-2) contains supplementary material, which is available to authorized users.

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