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.

Figure 1:. Index TMA layout for PD-L1 quantification.

(A) Schematic map along with ten standardized cell line codes and (B) representative image of the stained standardized TMA and 300um diameter ROIs as white circles.

Figure 2:. Overview of Experimental Plan.

Three standardized TMA were stained with Human IO panel and nuclear stain. First two experiments were stained and collected in the same week. Third experiment was stained and each row was collected one week apart.

Figure 3:. Correlation of Log₂ transformed PD-L1 data from GeoMx DSP, QIF and IHC DAB.

Regression of PD-L1 protein expression by DSP with (A-C) QIF assay performed using 3 antibodies (E1L3N, SP142, and SP263) and (D-H) IHC DAB assay performed using 5 antibodies (E1L3N, SP142, SP263, 22C3 and 28–8). Each dot represents average of 2 TMAs (GeoMx DSP), 3 TMAs (QIF) and 20 TMAs (IHC DAB) with 3 pellet per cell clone in one TMA.

Figure 4:. Reproducibility across rows and experiments stained and collected in the same week.

Bar graph show (A) signal to noise ratio and (B) PD-L1 counts for three rows from same experiment. Regression on Log₂ scale for PD-L1 counts (C) between two rows from same experiment and (D) average PD-L1 counts of three rows between two experiments.

Figure 5:. Reproducibility across rows collected one week apart from same staining.

(A) Distribution of PD-L1, Histone H3 and average IC raw counts from all cell pellet collected one week apart. Bar graph show (B) signal to noise ratio and (C) PD-L1 counts for three rows. Regression on Log₂ scale for PD-L1 counts (D-E) between two rows collected on different weeks.