Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer

Abstract

PD-L1 is expressed in a percentage of lung cancer patients and those patients show increased likelihood of response to PD-1 axis therapies. However, the methods and assays for the assessment of PD-L1 using immunohistochemistry are variable and PD-L1 expression appears to be highly heterogeneous. Here, we examine assay heterogeneity parameters toward the goal of determining variability of sampling and the variability due to pathologist-based reading of the immunohistochemistry slide. SP142, a rabbit monoclonal antibody, was used to detect PD-L1 by both chromogenic immunohistochemistry and quantitative immunofluorescence using a laboratory-derived test. Five pathologists scored the percentage of PD-L1 positivity in tumor- and stromal-immune cells of 35 resected non-small cell lung cancer cases, each represented on three separate blocks. An intraclass correlation coefficient of 94% agreement was seen among the pathologists for the assessment of PD-L1 in tumor cells, but only 27% agreement was seen in stromal/immune cell PD-L1 expression. The block-to-block reproducibility of each pathologist's score was 94% for tumor cells and 75% among stromal/immune cells. Lin's concordance correlation coefficient between pathologists' readings and the mean immunofluorescence score among blocks was 94% in tumor and 68% in stroma. Pathologists were highly concordant for PD-L1 tumor scoring, but not for stromal/immune cell scoring. Pathologist scores and immunofluorescence scores were concordant for tumor tissue, but not for stromal/immune cells. PD-L1 expression was similar among all the three blocks from each tumor, indicating that staining of one block is enough to represent the entire tumor and that the spatial distribution of heterogeneity of expression of PD-L1 is within the area represented in a single block. Future studies are needed to determine the minimum representative tumor area for PD-L1 assessment for response to therapy.

Figure 1. Consort Diagram

This study included resections of 35 non-small cell lung cancer tumors. Three quantitative immunofluorescence cases were rejected due to the technical artifact of antibody trapping.

Figure 2. Illustration of quantitative assessment of optimal titration

Quantitative assessment of optimal antibody titer is achieved by plotting the average of the top 10% of scores on the test tissue microarray (blue line) and the average of the bottom 10% of the scores (red line) for each antibody concentration tested (X axis). The optimal titration is the maximal signal to noise (shown on the right side Y axis) plotted (orange line) to show a peak at 0.154ug/ml.

Figure 3. Images and Heatmaps

Whole tissue sections cut from 3 separate blocks from the same case. The top 3 panels indicate PD-L1 diaminobenzidine staining among all 3 blocks. The bottom 3 panels show PD-L1 quantitative immunofluorescence staining of serial sections of corresponding blocks. The heatmaps are based on quantitative immunofluorescence data, which generated a quantitative immunofluorescence score as an arbitrary unit of fluorescence for each field of view within the tumor. The quantitative immunofluorescence score scale is presented below the heatmaps.

Figure 4. Distribution of Maximum PD-L1 score among 5 Pathologists

A) A histogram of all chromogenic immunohistochemistry data for tumor: the raw percentage of staining assigned by each of the 5 pathologists for each of the 3 blocks, per case (15 bars per case, color coded by block as shown in the inset). B) the distribution of the single maximum score provided by each of the 5 pathologists among all 3 blocks per case from tumor regions (5 data points per case). Each boxplot represents 25^th%-, median-, and 75^th% readings, with the whiskers denoting minimum and maximum percentages of these 5 data points. The y-axis labels the maximum reading among the 3 blocks. C) shows a histogram of all chromogenic immunohistochemistry data for stroma: the raw percentage of staining assigned by each of the 5 pathologists for each of the 3 blocks, per case (15 bars per case, color coded by block as shown in the inset). D) the distribution of the single maximum score provided by each of the 5 pathologists among all 3 blocks per case from stromal regions (5 data points per case). Each boxplot represents 25^th%-, median-, and 75^th% readings, with the whiskers denoting minimum and maximum percentages of these 5 data points. The y-axis labels the maximum reading among the 3 blocks.

Figure 5. Quantitative Immunofluorescence vs. Chromogenic Immunohistochemistry

The quantitative immunofluorescence score is shown as a box and whisker plot for representative fields of view from each case. Each box represents the 25^th%-, median-, and 75^th% quantitative immunofluorescence score of the respective case. Whiskers represent the minimum and maximum score. The X-axis indicates all cases organized by their median quantitative immunofluorescence score values. Cases are also color coded by their PD-L1 diaminobenzidine staining percentages: those in blue stained <1%, those in red stained 1–50%, and those in green stained >50%. A) the scores for the PD-L1 expression in the tumor. B) the scores for the PD-L1 expression in the stroma.

Figure 6. Regressions of Maximum and Mean quantitative immunofluorescence score vs. Maximum Pathologists’ Score

The maximum percentage PD-L1 staining among all pathologists was regressed with the maximum quantitative immunofluorescence score among all 3 blocks, in both A) tumor and B) stromal regions. Also, the maximum percentage PD-L1 staining among all pathologists was regressed with the highest average quantitative immunofluorescence score of a block among 3 blocks, in both C) tumor and D) stromal regions.