Figure 3. ANNUBP and Cluster 2 lesions are characterized by enhanced signatures of antigen presentation, immune surveillance and T cell infiltration.

(A) Volcano plots illustrating the top differentially expressed genes in Cluster 2 (n=10) versus Cluster 1 (n=14) lesions. The x-axis represents the log₂ fold change in gene expression, whereas the y-axis represents the −log₁₀ Benjamini Hochberg adjusted p-value. An adjusted p-value of 0.05 was set as the false discovery threshold as denoted by the dotted line. Genes with log₂ fold changes ≥1 are colored red, whereas those with log₂ fold changes ≤ −1 are colored blue. (B) Box and whisker plot of normalized antigen presentation signature scores in Cluster 1 (n=14), Cluster 2 (n=10), and Cluster 3 (n=11) tumors. Dots represent individual samples. Whiskers extend from the minima to maxima that are no further than 1.5x the inter-quartile range spanning the first to third quartiles. The center line represents the median. The box spans the 25^th to 75^th percentiles. Data beyond the whiskers are outliers and are plotted as individual points. P-values represent unpaired, two-tailed t-tests between groups as shown. (C) Box and whisker plots depicting HLA-DPA1 and HLA-DPB1 mRNA expression (normalized counts) stratified by molecular cluster. P-values represent unpaired, two-tailed t-tests between groups. (D) Volcano plot illustrating the top differentially expressed genes in Cluster 3 (n=11) vs Cluster 2 (n=10) lesions. (E) Volcano plot showing top differentially expressed genes in MPNST (n=6) vs ANNUBP (n=10). (F) Box and whisker plots depicting HLA-DQA1 and HLA-DQB1 mRNA expression (normalized counts) stratified by molecular cluster. (G) Representative photomicrographs of T cell subsets identified by immunofluorescence staining of CD4 (orange), CD8 (green) and FOXP3 (white) in PNF, ANNUBP and MPNST tissue sections. DAPI (blue) is shown as a nuclear counterstain. Magnification denoted by 100 μm scale bars with inset high-power magnification as shown. (H) Barplots reflecting quantitative analysis of T cell subsets normalized to the total number of nuclei per high power field (HPF). Error bars reflect standard error of the mean (SEM). PNF (n=6, ROI=30), ANNUBP (n=4, ROI=20) and MPNST (n=5, ROI=25) were analyzed by one-way ANOVA using Tukey’s multiple comparisons test. (I) Box and whisker plot of normalized cytotoxicity signature scores in Cluster 1 (n=14), Cluster 2 (n=10), and Cluster 3 (n=11) tumors. P-values represent unpaired, two-tailed t-tests between groups as shown. (J) Box and whisker plot depicting CTLA4 mRNA expression (normalized counts) stratified by molecular cluster. P-values represent unpaired, two-tailed t-tests between groups.