Digital quantitative assessment of PD-L1 using digital spatial profiling


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.

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Fig. 1: Index TMA layout for PD-L1 quantification.
Fig. 2: Overview of experimental plan.
Fig. 3: Correlation of Log2 transformed PD-L1 data from GeoMx DSP, QIF and IHC DAB.
Fig. 4: Reproducibility across rows and experiments stained and collected in the same week.
Fig. 5: Reproducibility across rows collected one week apart from same staining.


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This study was supported by instrumentation from NanoString and funding from the Breast Cancer Research Foundation and the Yale SPORE in Lung Cancer.

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DLR and SG conceived the study, supervised the analysis and revised the final version of the manuscript. SG and JZ selected the study specimens and carried out the GeoMx DSP data analysis. SG drafted the manuscript. KF performed the GeoMx DSP assay. SMM carried out fluorescent and chromogenic IHC assay. All authors have read and approved the final version of the manuscript.

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Correspondence to David L. Rimm.

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Conflict of interest

DLR is a consultant/advisor to Amgen, Astra Zeneca, Agendia, Biocept, BMS, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GSK, InVicro/Konica Minolta, Lilly, Merck, Perkin Elmer, PAIGE.AI, Sanofi and Ultivue. KF is an employee of Nanostring. The remaining authors declare no competing interests.

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Gupta, S., Zugazagoitia, J., Martinez-Morilla, S. et al. Digital quantitative assessment of PD-L1 using digital spatial profiling. Lab Invest 100, 1311–1317 (2020).

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