Table 1.
Patient’s clinical information.
Table 2.
Fig 1.
Example of computational tissue analysis (cTA) of NSCLC sections.
A: Manual inclusion (green line) and exclusion (red line) annotations in squamous cell carcinoma tissue for CD56 IHC. B: Enlarged view area from (A) showing exclusion annotation around a central necrotic area in tumor-nest (arrow) and a segment of the inclusion annotation around the tumor mass margin (arrowhead). C: PD-L1 IHC of a squamous cell carcinoma. D: Algorithm markup from (C) showing membrane scoring only in cells assigned to the tumor-nest compartment, with 1+ = yellow, 2+ = orange, and 3+ = red. The separated TME compartment cell nuclei are marked in green. Scale bars: 1 mm (A); 100 μm (B); and 150 μm (C and D). NSCLC = non-small cell lung carcinoma; IHC = immunohistochemistry; TME = tumor microenvironment.
Table 3.
Classification of NSCLC according to PD-L1 expression patterns in tumor-nest and TME compartments.
Fig 2.
Relationships between biomarkers in our NSCLC cohort.
(A) Hierarchical clustering heat map of percentages of positive cells for FOXP3, PD-L1, CD8, Granzyme B, and CD68. Each biomarker (row) was normalized before clustering to give a mean of 0 and a standard deviation of 1 across columns (samples). The color scale indicates the relative size of the biomarker score compared to the other samples in the cohort with blue = low, white = neutral, and brown = high. Bracket height indicates the degree of correlation between measurements with shorter brackets representing higher correlations and taller brackets representing smaller correlations. (B) Heatmap of Pearson correlation coefficients between percent positive cells measurements across samples for FOXP3, PD-L1, CD8, Granzyme B, and CD68. The color scale ranges from the minimum (blue) and maximum (red) correlation coefficient observed in this analysis. Therefore red represents strong positive correlations, white represents moderate correlations, and blue represents weak correlations. No strong negative correlations were observed.
Table 4.
Average percent positive cells for multiple immune biomarkers in NSCLC tissue compartments.
Fig 3.
Immune markers in NSCLC relative to a 50% cut-off point for PD-L1 staining.
A: Percentage of positive cells for each biomarker in “All Cells” (tumor nest and TME compartments combined) calculated by using Computational Tissue Analysis (cTA™). NSCLC with >50% PD-L1-positive tumor cells were compared with those with lower expression. Statistical differences were calculated using two-tailed Student’s t-tests, assuming unequal sample variances (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001). Percentages of cells positive for CD68, CD8, FoxP3, and Granzyme B were significantly higher in the >50% PD-L1 group. B: Representative IHC images of PD-L1-, CD8-, and CD68-positive cells (the most numerous biomarkers) in an NSCLC (solid carcinoma) case with >50% PD-L1 expression in the tumor nest compartment, and an NSCLC (adenocarcinoma with mixed subtypes) case with lower PD-L1 expression. Scale bars = 100 μm. NSCLC = non-small cell lung carcinoma; IHC = immunohistochemistry; TME = tumor microenvironment.
Fig 4.
Examples of PD-L1 staining patterns observed and categorized in this NSCLC series.
A: Pattern I—Constitutive mixed with induced. Diffuse expression of PD-L1 (IHC) on tumor cell membranes of a squamous cell carcinoma, including central regions of trabeculae. Prominent labeling of cells in the TME compartment at the tumor-nest-TME interface suggesting presence of an immunological synapse (inset arrow). B: Pattern II—Induced only. Patchy expression of PD-L1 in a squamous cell carcinoma at the tumor-nest-TME interface (inset arrow). Minimal to no PD-L1 expression in the trabeculae (asterisk) if compared with (A). C: Pattern III—Immune ignorance. No to minimal PD-L1 expression in both tumor and TME compartments in an adenocarcinoma. D: Pattern IV—Constitutive only. Diffuse expression of PD-L1 by tumor-nests in an adenocarcinoma with minimal TME staining. E: Pattern V—TME expression only. No to minimal PD-L1 expression in tumor cells of a squamous cell carcinoma, with widespread staining in the TME compartment. F: Percentages of PD-L1-positive tumor cells in all staining pattern categories (I-V). G: Percentages of TME PD-L1-positive cells in all staining pattern categories (I-V). H: Digital H-scores from tumor-nests in all staining pattern categories (I-V). Significant differences between groups I and II were calculated using two-tailed Student’s t-tests, assuming unequal sample variances (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001). Scale bars: 200 μm (A-E). NSCLC = non-small cell lung carcinoma; IHC = immunohistochemistry; TME = tumor microenvironment.