Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray.
ABSTRACT: Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.
Project description:Programmed death ligand-1 (PD-L1) expression as determined by immunohistochemistry (IHC) is potentially predictive of clinical outcome. The aim of this study was to assess the concordance of reported PD-L1 IHC assays and investigate factors influencing variability. Consecutive sections from 20 non-small cell lung cancers (NSCLCs) comprising resection, core biopsy, cytology and pleural fluid samples underwent IHC with 5 different antibody/autostainer combinations: 22C3/Link48, 28-8/BOND-MAX, E1L3N/BOND-MAX, SP142/BenchMark and SP263/BenchMark. PD-L1 RNA levels were assessed using RNAscope. The frequency of positive cases using scoring thresholds from clinical trials was 72%, 33%, 61%, 56%, and 33% for the 5 IHC protocols respectively, and 33% for RNAscope. Pairwise agreement on the classification of cases as positive or negative for PD-L1 expression ranged from 61%-94%. On a continuous scale, the lowest correlation was between 28-8/BOND-MAX and SP142/BenchMark (R2=0.25) and highest was between 22C3/Link48 and E1L3N/BOND-MAX (R2=0.71). When cases were ordered according to tumor cell (TC)%, a similar ranking of cases across IHC protocols could be observed, albeit with different quanta and limits of detection. Single-slide OPAL 7-color fluorescence IHC analysis revealed a high degree of co-localization of staining from the 5 PD-L1 antibodies. Using SP142 antibody in a BOND-MAX protocol led to increased TC% quanta, while retaining a similar ranking of samples according to TC%. The results of this study highlight tumor PD-L1 status can vary significantly according to IHC protocol. Protocol-dependent staining intensities and nominated thresholds for positivity contribute to this variability, while the antibody used appears to be less of a factor.
Project description:Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) assays are widely used for complementary or companion diagnostic purposes during treatment with immune checkpoint inhibitors. However, limited information is available on the clinical reliability of the PD-L1 IHC assay using small biopsy samples.Participants included 46 patients with nonsmall cell lung cancer who underwent PD-L1 testing using 3 PD-L1 IHC assays (22C3, SP142, and SP263) for both small biopsy samples and surgical specimens from November 2017 to June 2018. The PD-L1 IHC assay results were analyzed with cut-off values of 1%, 5%, 10%, and 50%. The PD-L1 IHC results obtained from the surgical specimens were regarded as the reference values.The 22C3, SP142, and SP263 PD-L1 IHC assays were performed in 26 (57%), 20 (43%), and 46 (100%) patients, respectively. Biopsy methods included radial probe endobronchial ultrasound using a guide sheath, endobronchial ultrasound-guided transbronchial needle aspiration, bronchoscopic biopsy, and percutaneous needle aspiration in 26 (57%), 4 (9%), 12 (25%), and 4 (9%) patients, respectively. The 22C3, SP142, and SP263 PD-L1 assays had concordance rates of 73-96, 65-80, and 72%-91%, respectively, compared with the reference values.PD-L1 testing with 3 commercial PD-L1 IHC assays using small biopsy samples is reliable in patients with nonsmall cell lung cancer.
Project description:Programmed death ligand-1 (PD-L1) immunohistochemistry is used to guide treatment decisions regarding the use of checkpoint immunotherapy in the management of urothelial carcinoma of the bladder and hypopharyngeal (HP) squamous cell carcinoma. With increasing PD-L1 testing options, a need has arisen to assess the analytical comparability of diagnostic assays in order to develop a more sustainable testing strategy. Using tissue microarrays, PD-L1 expression in tumor cells (TCs) and immune cells (ICs) was manually scored in 197 cases and 27 cases of bladder and HP cancer, respectively. Three commercial kits (Ventana SP263, Ventana SP142, Dako 22C3) and 1 platform-independent test (Cell Signalling Technologies E1L3N) were utilized. Across the 3 commercially available clones, 14% and 74% of urothelial carcinomas were positive and negative, respectively, whereas 7% and 78% of HP carcinomas were positive and negative, respectively. Twelve percent of bladder and 15% HP cases showed discrepant PD-L1 classification results. Regardless of the scoring algorithm used, E1L3N provided comparable PD-L1 staining results. Fleiss' kappa and intraclass correlation coefficient (ICC) analyses demonstrated substantial agreement among all antibody clones (k=0.639 to 0.791) and excellent reliability among SP263, 22C3, and E1L3N antibodies (ICC, 0.929 to 0.949) in TC staining. Compared with the other 3 clones, SP142 TC staining was lower with only moderate correlation (ICC, 0.500 to 0.619). Generally, the reliability of immune cell staining was lower compared with TC staining (ICC, 0.519 to 0.866). Our results demonstrate good analytic comparability of all 4 antibodies. The results are encouraging and support growing optimism in the pathology and oncology communities concerning strategies in PD-L1 assay use.
Project description:The expression of programmed cell death ligand 1 (PD-L1) is a biomarker for immunotherapy, but approved detection method is absent in diffuse large B-cell lymphoma (DLBCL). Here, we performed three methods including immunohistochemistry (IHC) (clone SP263 and SP142), RNAscope, and fluorescence in situ hybridization (FISH) to evaluate PD-L1 status on a cohort of DLBCL including 94 of DLBCL-NOS, 25 of primary mediastinal large B-cell lymphoma (PMBCL) and 7 of double-hit lymphoma (DHL). SP263 with 25% for immune cell (IC) or combined cell and SP142 with 10% for tumor cell (TC), 20% for both of IC and combined cell were proved to have corresponding survival prognostic. Combined<sup>+</sup> showed comparable prognostic value with TC<sup>+</sup> and IC<sup>+</sup> . SP263 and SP142 showed strong concordance (k = 0.788) with combined<sup>+</sup> rates of 33.3% (42/126) and 34.9% (44/126), respectively. In DLBCL-NOS, TC<sup>+</sup> by SP263 preferred to non-GCB and immunoblastic variant DLBCL-NOS (P = 0.029 and P = 0.004). Combined<sup>+</sup> (SP263 and SP142) were associated with more than one extranodal site involved (P = 0.006, P = 0.042), higher ECOG PS scores (P = 0.001, P < 0.001), high IPI risk (P = 0.012, P = 0.005), and poor treatment response (P = 0.095, P = 0.002). IC<sup>+</sup> by SP263 and SP142 were both independent risk factors (P = 0.027, P = 0.037). 9p24.1 locus amplification and gain were identified in 4.3% and 7.6% DLBCL-NOS and indicated shorter overall survival (P = 0.004). Positive rate of PD-L1 by RNAscope was 36.5%, while no clinical significance shown. PD-L1 positive rates were all higher in PMBCL and DHL than in DLBCL-NOS by SP263, SP142, RNAscope, and FISH (P = 0.001, P < 0.001, P = 0.005 and P < 0.001, respectively). In conclusion, combined PD-L1 expression by IHC was potentially reliable and convenient as a predicting biomarker. SP263 staining was easier to evaluate and recognized more PD-L1-stained cells, but SP142 presented a better prognostic indicator. FISH and RNAscope could be used as supplementary assays. PMBCL itself was a sensitive cohort for immunotherapy.
Project description:BACKGROUND:Antibodies targeting the programmed cell death-1 (PD-1)/PD-ligand 1 (PD-1/PD-L1) checkpoint have shown promising clinical activity in patients with advanced urothelial carcinoma (UC). Expression of PD-L1 in UC tumors has been investigated using different antibody clones, staining protocols, and scoring algorithms. The aim was to establish the extent of concordance among PD-L1 immunohistochemistry (IHC) assays. METHODS:Tumor biopsy samples (N = 335) were assessed using four commercially available PD-L1 assays: VENTANA SP263, VENTANA SP142, PD-L1 IHC 28-8 pharmDx, and PD-L1 IHC 22C3 pharmDx. PD-L1 analytical staining and classification concordance, including agreement between clinically relevant scoring algorithms, were investigated using overall/positive/negative percentage agreement (OPA/PPA/NPA). RESULTS:Good analytical correlation was observed among the VENTANA SP263, PD-L1 IHC 22C3 pharmDx, and PD-L1 IHC 28-8 pharmDx assays for tumor cell (TC) and immune cell (IC) PD-L1 staining with Spearman rank coefficients of 0.92-0.93 for TCs and 0.88-0.91 for ICs. However, concordance (preset criterion: ≥85%) between patient PD-L1 status when applying the TC or ICICArea ≥ 25% (VENTANA SP263) cutoff was only achieved for PD-L1 IHC 22C3 pharmDx versus VENTANA SP263 (OPA 92.2%, PPA 86.4%, NPA 95.4%). Differences were observed between patient populations with UC tumors classified as PD-L1 high versus PD-L1 low/negative using combined positive score (CPS) ≥1, CPS ≥10, IC ≥5%, and TC/IC ≥25%. CONCLUSIONS:The VENTANA SP263 and PD-L1 IHC 22C3 pharmDx assays are analytically similar in UC. When the different PD-L1 assays were combined with their specified clinical scoring algorithms, differences were seen in patient classification driven by substantial differences in scoring approaches.
Project description:BACKGROUND:Immunohistochemistry (IHC) for programmed cell death ligand 1 (PD-L1) displays staining diversity. We compared IHC staining of PD-L1 in gastric cancer (GC) by using three commercially available antibody clones, and analyzed the correlation with the prognosis. METHODS:IHC using PD-L1 antibodies (clones SP142, 28-8 and E1L3N) in 315 formalin-fixed paraffin-embedded samples was qualitatively compared at the 1, 5 and 10% cut-off by two pathologists on total, tumor and immune/stromal cells. We used computer - assisted scoring to quantitatively analyze and compare the "H-score" of PD-L1 expression in 66 samples on total cells. The antibody clone SP142 was selected to investigate the infiltration of PD-L1+CD8+ T cells using automated quantitative immunofluorescence analyses (n?=?50) and the prognostic significance. The prognoses were assessed by log-rank test. RESULTS:PD-L1 clones SP142 and 28-8 displayed great concordance by qualitative (??=?0.816, 0.810 for total cells and tumor cells at the 5% cut-off) and quantitative analyses (R2?=?0.7991, 0.8187 for positive percentage and "H-score"). PD-L1 clone SP142 showed the highest positivity in immune/stromal cells staining (18.41%) compared to 28-8 (7.62%), while clone E1L3N showed poor staining in both tumor and immune/stromal cells. Clone SP142, but not 28-8 and E1L3N, predicted a worse prognosis at the 5% cut-off (p?=?0.0243). Both the clone SP142 and 28-8 had high inter-pathologist correlation for tumor staining (R2?=?0.9805 and R2?=?0.9853), but a moderate correlation for stromal/immune cell staining (R2?=?0.5653 and R2?=?0.5745). Furthermore, a higher density of PD-L1+CD8+ T cells was correlated with a shorter survival time (R2?=?0.0909, p?=?0.0352). CONCLUSIONS:PD-L1 antibody clone SP142 was superior in cell staining, particularly in immune/stromal cell and prognosis. These findings are important for selection of PD-L1 antibody clones in the future diagnostic test.
Project description:Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression.To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies.PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined.PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen ? range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02).Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.
Project description:Purpose:Triple-negative breast cancer (TNBC) represents a major clinical challenge due to its aggressive and metastatic behavior and the lack of available targeted therapies. Therefore, therapeutic strategies are needed to improve TNBC patient management. Recently, atezolizumab and nab-paclitaxel chemotherapy has been approved by the Food and Drug Administration for the first-line treatment of patients with locally advanced and metastatic TNBC. The programmed death-ligand 1 (PD-L1) immunohistochemical SP142 assay was also approved as a companion diagnostic device for selecting TNBC patients for atezolizumab treatment. This study aimed to evaluate and compare the analytical performance of the PD-L1 22C3/SP263 assays in comparison with the SP142 assay for ? 1% immune cells (ICs). Methods:Immunohistochemical expression for the PD-L1 22C3/SP263 assays, in comparison with the SP142 assay, was analyzed for the ? 1% ICs in 95 TNBCs. Results:At the 1% cut-off value, the proportions of positive cases were 52.6% for the SP142 assay in infiltrating ICs and 50.5% and 52.6% for the 22C3 and SP263 assays in tumor cells, respectively. The PD-L1 SP263 assay had the highest while the PD-L1 22C3 assay had the lowest total positive expression rate at all cut-off values. The concordance rate between the assays was highest at a 1% cut-off value and decreased when the cut-off value increased. The concordance rate between the SP142 and SP263 assays at 1% cut-off was high, while in comparison, the concordance rate between the SP142 and 22C3 assays at 1% cut-off was relatively lower. Conclusion:This study demonstrates that although the 22C3 assay at a 1% cut-off value compared with the PD-L1 SP142 assay at the clinically relevant cut-off shows comparable but not interchangeable analytical performance, the analytical performance of the SP263 assay at a 1% cut-off value shows interchangeable performance with the PD-L1 SP142 assay at the clinically relevant cut-off.
Project description:Importance:Four assays registered with the US Food and Drug Administration (FDA) detect programmed cell death ligand 1 (PD-L1) to enrich for patient response to anti-programmed cell death 1 and anti-PD-L1 therapies. The tests use 4 separate PD-L1 antibodies on 2 separate staining platforms and have their own scoring systems, which raises questions about their similarity and the potential interchangeability of the tests. Objective:To compare the performance of 4 PD-L1 platforms, including 2 FDA-cleared assays, 1 test for investigational use only, and 1 laboratory-developed test. Design, Setting, and Participants:Four serial histologic sections from 90 archival non-small cell lung cancers from January 1, 2008, to December 31, 2010, were distributed to 3 sites that performed the following immunohistochemical assays: 28-8 antibody on the Dako Link 48 platform, 22c3 antibody on the Dako Link 48 platform, SP142 antibody on the Ventana Benchmark platform, and E1L3N antibody on the Leica Bond platform. The slides were scanned and scored by 13 pathologists who estimated the percentage of malignant and immune cells expressing PD-L1. Statistical analyses were performed from December 1, 2015, to August 30, 2016, to compare antibodies and pathologists' scoring of tumor and immune cells. Main Outcomes and Measures:Percentages of malignant and immune cells expressing PD-L1. Results:Among the 90 samples, the SP142 assay was an outlier, with a significantly lower mean score of PD-L1 expression in both tumor and immune cells (tumor cells: 22c3, 2.96; 28-8, 3.26; SP142, 1.99; E1L3N, 3.20; overall mean, 2.85; and immune cells: 22c3, 2.15; 28-8, 2.28; SP142, 1.62; E1L3N, 2.28; overall mean, 2.08). Pairwise comparisons showed that the scores from the 28-8 and E1L3N tests were not significantly different but that the 22c3 test showed a slight (mean difference, 0.24-0.30) but statistically significant reduction in labeling of PD-L1 expression in tumor cells. Evaluation of intraclass correlation coefficients (ICCs) between antibodies to quantify interassay variability for PD-L1 expression in tumor cells showed high concordance between antibodies for tumor cell scoring (0.813; 95% CI, 0.815-0.839) and lower levels of concordance for immune cell scoring (0.277; 95% CI, 0.222-0.334). When examining variability between pathologists for any single assay, the concordance between pathologists' scoring for PD-L1 expression in tumor cells ranged from ICCs of 0.832 (95% CI, 0.820-0.844) to 0.882 (95% CI, 0.873-0.891) for each assay, while the ICCs from immune cells for each assay ranged from 0.172 (95% CI, 0.156-0.189) to 0.229 (95% CI, 0.211-0.248). Conclusions and Relevance:The assay using the SP142 antibody is an outlier that detected significantly less PD-L1 expression in tumor cells and immune cells. The assay for antibody 22c3 showed slight yet statistically significantly lower staining than either 28-8 or E1L3N, but this significance was detected only when using the mean of 13 pathologists' scores. The pathologists showed excellent concordance when scoring tumor cells stained with any antibody but poor concordance for scoring immune cells stained with any antibody. Thus, for tumor cell assessment of PD-L1, 3 of the 4 tests are concordant and reproducible as read by pathologists.