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Digital quantitative assessment of PD-L1 using digital spatial profiling.

ABSTRACT: The assessment of programmed death 1 ligand 1 (PD-L1) expression by Immunohistochemistry (IHC) is the US Food and Drug Administration (FDA)-approved predictive marker to select responders to checkpoint blockade anti-PD-1/PD-L1 axis immunotherapies. Different PD-L1 immunohistochemistry (IHC) assays use different antibodies and different scoring methods in tumor cells and immune cells. Multiple studies have compared the performance of these assays with variable results. Here, we investigate an alternative method for assessment of PD-L1 using a new technology known as digital spatial profiling. We use a previously described standardization tissue microarray (TMA) to assess the accuracy of the method and compare digital spatial profiler (DSP) to each FDA-approved PD-L1 assays, one LDT assay and three quantitative fluorescence assays. The standardized cell line Index tissue microarray contains 10 isogenic cells lines in triplicates expressing various ranges of PD-L1. The dynamic range of PD-L1 digital counts was measured in the ten cell lines on the Index TMA using the GeoMx DSP assay and read on the nCounter platform. The digital method shows very high correlation with immunohistochemistry scored with quantitative software and with quantitative fluorescence. High correlation of PD-L1 digital DSP counts were seen between rows on the same Index TMA. Finally, experiments from two Index TMAs showed reproducibility of DSP counts were independent of variable slide storage time over a three-week period after antibody labeling but before collection of cleaved tags. In summary, DSP appears to have quantitative potential comparable to quantitative immunohistochemistry. It is possible that this technology could be used as a PD-L1 protein measurement system for companion diagnostic testing for immune therapy.

SUBMITTER: Gupta S

PROVIDER: S-EPMC7502436 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Digital quantitative assessment of PD-L1 using digital spatial profiling.

Gupta Swati S Zugazagoitia Jon J Martinez-Morilla Sandra S Fuhrman Kit K Rimm David L DL

Laboratory investigation; a journal of technical methods and pathology 20200406 10

The assessment of programmed death 1 ligand 1 (PD-L1) expression by Immunohistochemistry (IHC) is the US Food and Drug Administration (FDA)-approved predictive marker to select responders to checkpoint blockade anti-PD-1/PD-L1 axis immunotherapies. Different PD-L1 immunohistochemistry (IHC) assays use different antibodies and different scoring methods in tumor cells and immune cells. Multiple studies have compared the performance of these assays with variable results. Here, we investigate an alt ...[more]

PMID: 32249818

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Project description: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:PurposeDespite remarkable clinical responses and prolonged survival across several cancers, not all patients benefit from PD-1/PD-L1 immune checkpoint blockade. Accordingly, assessment of tumour PD-L1 expression by immunohistochemistry (IHC) is increasingly applied to guide patient selection, therapeutic monitoring, and improve overall response rates. However, tissue-based methods are invasive and prone to sampling error. We therefore developed a PET radiotracer to specifically detect PD-L1 expression in a non-invasive manner, which could be of diagnostic and predictive value.MethodsAnti-PD-L1 (clone 6E11, Genentech) was site-specifically conjugated with DIBO-DFO and radiolabelled with 89Zr (89Zr-DFO-6E11). 89Zr-DFO-6E11 was optimized in vivo by longitudinal PET imaging and dose escalation with excess unlabelled 6E11 in HCC827 tumour-bearing mice. Specificity of 89Zr-DFO-6E11 was evaluated in NSCLC xenografts and syngeneic tumour models with different levels of PD-L1 expression. In vivo imaging data was supported by ex vivo biodistribution, flow cytometry, and IHC. To evaluate the predictive value of 89Zr-DFO-6E11 PET imaging, CT26 tumour-bearing mice were subjected to external radiation therapy (XRT) in combination with PD-L1 blockade.Results89Zr-DFO-6E11 was successfully labelled with a high radiochemical purity. The HCC827 tumours and lymphoid tissue were identified by 89Zr-DFO-6E11 PET imaging, and co-injection with 6E11 increased the relative tumour uptake and decreased the splenic uptake. 89Zr-DFO-6E11 detected the differences in PD-L1 expression among tumour models as evaluated by ex vivo methods. 89Zr-DFO-6E11 quantified the increase in PD-L1 expression in tumours and spleens of irradiated mice. XRT and anti-PD-L1 therapy effectively inhibited tumour growth in CT26 tumour-bearing mice (p < 0.01), and the maximum 89Zr-DFO-6E11 tumour-to-muscle ratio correlated with response to therapy (p = 0.0252).ConclusionPET imaging with 89Zr-DFO-6E11 is an attractive approach for specific, non-invasive, whole-body visualization of PD-L1 expression. PD-L1 expression can be modulated by radiotherapy regimens and 89Zr-DFO-6E11 PET is able to monitor these changes and predict the response to therapy in an immunocompetent tumour model.

Project description:ImportanceEarly-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.ObjectiveTo 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.Design, setting, and participantsPD-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.Main outcomes and measuresPD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.ResultsUsing chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ 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).Conclusions and relevanceObjective 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.

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Digital quantitative assessment of PD-L1 using digital spatial profiling.

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Digital quantitative assessment of PD-L1 using digital spatial profiling.

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