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.

Introduction:In the phase IV, open-label, single-arm study NCT01203917, first-line gefitinib 250 mg/d was effective and well tolerated in Caucasian patients with epidermal growth factor receptor (EGFR) mutation-positive non–small-cell lung cancer (previously published). Here, we report EGFR mutation analyses of plasma-derived, circulating-free tumor DNA.Methods:Mandatory tumor and duplicate plasma (1 and 2) baseline samples were collected (all screened patients; n = 1060). Preplanned, exploratory analyses included EGFR mutation (and subtype) status of tumor versus plasma and between plasma samples. Post hoc, exploratory analyses included efficacy by tumor and plasma EGFR mutation (and subtype) status.Results:Available baseline tumor samples were 1033 of 1060 (118 positive of 859 mutation status known; mutation frequency, 13.7%). Available plasma 1 samples were 803 of 1060 (82 positive of 784 mutation status known; mutation frequency, 10.5%). Mutation status concordance between 652 matched tumor and plasma 1 samples was 94.3% (95% confidence interval [CI], 92.3–96.0) (comparable for mutation subtypes); test sensitivity was 65.7% (95% CI, 55.8–74.7); and test specificity was 99.8% (95% CI, 99.0–100.0). Twelve patients of unknown tumor mutation status were subsequently identified as plasma mutation-positive. Available plasma 2 samples were 803 of 1060 (65 positive of 224 mutation status-evaluable and -known). Mutation status concordance between 224 matched duplicate plasma 1 and 2 samples was 96.9% (95% CI, 93.7–98.7). Objective response rates are as follows: mutation-positive tumor, 70% (95% CI, 60.5–77.7); mutation-positive tumor and plasma 1, 76.9% (95% CI, 65.4–85.5); and mutation-positive tumor and mutation-negative plasma 1, 59.5% (95% CI, 43.5–73.7). Median progression-free survival (months) was 9.7 (95% CI, 8.5–11.0; 61 events) for mutation-positive tumor and 10.2 (95% CI, 8.5–12.5; 36 events) for mutation-positive tumor and plasma 1.Conclusion:The high concordance, specificity, and sensitivity demonstrate that EGFR mutation status can be accurately assessed using circulating-free tumor DNA. Although encouraging and suggesting that plasma is a suitable substitute for mutation analysis, tumor tissue should remain the preferred sample type when available.

Purpose: Immunotherapies targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) demonstrate encouraging antitumor activity and manageable tolerability in non–small cell lung cancer (NSCLC), especially in patients with high tumor PD-L1 expression, as detected by companion or complementary diagnostic assays developed for individual agents. A laboratory is unlikely to use multiple assay platforms. Furthermore, commercially available diagnostic assays are not standardized, and different assay methods could lead to inappropriate treatment selection. This study establishes the extent of concordance between three validated, commercially available PD-L1 IHC diagnostic assays for NSCLC patients [Ventana SP263 (durvalumab), Dako 22C3 (pembrolizumab), and Dako 28-8 (nivolumab)]. Experimental Design: Five hundred formalin-fixed, paraffin-embedded archival NSCLC samples were obtained from commercial sources. Stained slides were read in batches on an assay-by-assay basis by a single pathologist trained in all methods, in a Clinical Laboratory Improvements Amendments program–certified laboratory. An additional pathologist performed an independent review of 200 stained samples for each assay. Results: PD-L1 expression was evaluable with all assays in 493 samples. The three assays showed similar patterns of tumor membrane staining, with high correlation between percent PD-L1 staining. An overall percentage agreement of >90% was achieved between assays at multiple expression cutoffs, including 1%, 10%, 25%, and 50% tumor membrane staining. Conclusions: This study builds optimism that harmonization between assays may be possible, and that the three assays studied could potentially be used interchangeably to identify patients most likely to respond to anti-PD-1/PD-L1 immunotherapies, provided the appropriate clinically defined algorithm and agent are always linked. Clin Cancer Res; 23(14); 3585–91. ©2017 AACR.

Tumors can evade immune detection by exploiting inhibitory immune checkpoints such as the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway. Antibodies that block this pathway offer a promising new approach to treatment in advanced/metastatic non-small cell lung cancer (NSCLC). A systematic review of the literature was conducted to assess the association of PD-L1 with important patient and disease characteristics, the prognostic significance of PD-L1 expressing NSCLC tumors, and the value of PD-L1 as a predictive biomarker of response to anti-PD-1/PD-L1 treatments in advanced/metastatic NSCLC. A total of 35 eligible studies were selected for analysis. Methods used to determine PD-L1 in NSCLC tissue varied considerably; with different PD-L1 antibodies, antibody detection methods, and staining cut-offs. Immunohistochemistry was the most frequent type of PD-L1 assay. Overall, study evidence did not support an association between PD-L1 expression and gender, age, smoking history, tumor histology (adenocarcinoma vs. squamous cell carcinoma), performance status, pathologic tumor grade or EGFR/KRAS/ALK mutational status. In several studies, high PD-L1 expression was associated with shorter survival compared with low expression. Most evidence indicated that patients with high vs. low PD-L1 expression were more likely to experience treatment benefit with anti-PD-1/PD-L1 agents (nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab) in advanced NSCLC. Variability in the methods used to determine PD-L1 expression in NSCLC tissue suggests a need for standardized use of well-validated PD-L1 diagnostic assays. Although considerable research links PD-L1 expression in tumors to shorter survival in advanced/metastatic NSCLC, its use as a prognostic factor requires more study. As studies of anti-PD-1/PD-L1 agents continue, PD-L1 is likely to play an important role as a predictive biomarker for selecting patients deriving most benefit from anti-PD-1/PD-L1 monotherapy and directing patients with lower levels of tumor PD-L1 expression (with a high unmet medical need), to alternative treatments, such as combination immunotherapies.

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