Fig. 7. Characterization of immune infiltration in AMPAC.

a Heatmap illustrated the characterization of three immune clusters. Each column represents a patient sample and rows indicate xCell signatures. The color of each cell shows the z-score of the xCell signature in that sample. AMPAC pathology classification, clinical features, and CNV status are shown above the heatmap. The χ² test was used to evaluate the association of pathology subtypes with the variables on the heatmap, and p values (****p < 1.0e−4, ***p < 1.0e−3, **p < 1.0e−2, *p < 0.05, n.s. > 0.05) were listed on the right. Single-sample Gene Set Enrichment Analysis (ssGSEA) based on proteomic and transcriptomic data were also applied to identify the dominant pathway signatures in each immune cluster. b Contour plot of two-dimensional density based on immune scores (y-axis) and stromal scores (x-axis) for different immune clusters. For each immune cluster, key upregulated pathways and molecules were reported based on RNA-seq (R), and proteomics (P) in the annotation boxes. c Kaplan–Meier curves for overall survival based on immune clusters (log-rank test, p = 0.048 for the comparison of 3 immune clusters, p = 0.16 for the comparison of M1 and M2, p = 0.18 for the comparison of M2 and M3, p = 0.013 for the comparison of M2 and M3). d The boxplot indicated immune scores among the three immune clusters (Kruskal–Wallis test, p < 1e−4). e Kaplan–Meier curves for overall survival based on immune score (log-rank test, p = 0.04). f The scatter plot described the correlation between the immune score and the xCell signatures (Spearman correlation). g The scatter plot described the correlation between the immune score and all focal events copy number (Spearman correlation). h Volcano plot showing the cis effect genes on 5q22.1 (Spearman correlation). i The top panel was 5q22.1 deletion focal event distribution, and the medium panel was the immune cluster distribution; the bottom heatmap showed immune score, 5q22.1 copy number, CAMK4 copy number, CAMK4 protein expression abundance, and CAMK4 mRNA abundance. j Kaplan–Meier curves for overall survival based on CAMK4 protein expression (log-rank test, p < 1e−4). k The scatter plot described the correlation between the CAMK4 kinase activity and substrates of CAMK4 (Spearman correlation). l Spearman correlation of CREB1 TF activity and expression of CREB1/S142 (Spearman’s r = 0.56, p = 0.049). m The scatter plot described the correlation between the CAMK4 TF activity and target genes of CAMK4 (Spearman correlation). n Kaplan–Meier curves for overall survival based on IL-6 mRNA expression (log-rank test, p = 0.014). o The schematic diagram summarized that CAMK4 cis effect changed its downstream signaling, and led to alterations in the immune microenvironment, which were associated with a poorer prognosis for patients.