Fig. 4: Genes differentially regulated by pembrolizumab treatment in treatment sensitive and resistant tumors.

A Heatmap of differentially regulated genes by pembrolizumab treatment to differentiate treatment sensitive from insensitive tumors. Samples are grouped by HS/CB or LS and sorted by hierarchical clustering within each group based on the similarity of change in gene-expression of selected genes. Genes are organized into four groups according to the combination of gene-regulation and differences between the two comparison groups. From top to bottom: genes up-regulated in both groups with higher expression in the HS/CB group, genes up-regulated in both groups with higher expression in the LS group, genes down-regulated in both groups with a greater degree of down-regulation in LS, and genes down-regulated in both groups with a greater degree of down-regulation in HS/CB. Genes in each group are listed in decreasing order of the difference of median change in gene-expression between HS/CB and LS groups. B Volcano plots of differentially expressed genes comparing paired early on-treatment to baseline tumor gene-expression in HS/CB or LS groups. Statistical significance for gene selection: FDR-adjusted p-value ≤ 0.10. C Distributions of change in gene-expression of immune checkpoint genes (TIGIT, PDCD1, CTLA4) in HS/CB (n = 11) versus LS (n = 11) baseline and on-treatment tumor pairs. The median for each group is marked by a horizontal line in the center of a rectangle and data points are shown as open circles. The distance between the third-quartile (Q3) and first-quartile (Q1), known as the interquartile range (IQR), is marked around the median by a rectangle. Vertical lines extending from the top and bottom of the rectangle show the maximum (Q3 + 1.5-times IQR) and minimum (Q1 + 1.5-times IQR). D PLA2G2D gene expression before and after pembrolizumab treatment in HS/CB (n = 11, p = 0.001) or LS (n = 11, p = 0.005) tumors before and after 2–3 cycles of pembrolizumab treatment. P-values were calculated by two-sided Wilcoxon rank-sum tests and uncorrected for multiple testing. The median for each group is marked by a horizontal line in the center of a rectangle and data points are shown as open circles. The distance between the third-quartile (Q3) and first-quartile (Q1), known as the interquartile range (IQR), is marked around the median by a rectangle. Vertical lines extending from the top and bottom of the rectangle show the maximum (Q3 + 1.5-times IQR) and minimum(Q1 + 1.5-times IQR). Source data are provided in SourceData_Fig4.zip. LS low sensitivity, HS/CB high-sensitivity/clinical benefit, FDR false discovery rate.