Fig. 7: Analysis of biomarkers for immunotherapy.

A Expression matrix based on log TPM values of selected immune checkpoints. The checkpoints were annotated on the left as co-inhibitory or co-stimulatory. The samples were annotated based on the tumor entity and the immune-cluster assignment. VTCN1 was highlighted in red. B Boxplot of VTCN1 expression (here vst values) in ACC1 (n = 25, median = 14.60, iqr = 1.16, max = 16.3, min = 6.19), ACC2 (n = 32, median = 14.10, iqr = 0.75, max = 15.5, min = 11.5), and non-ACC (n = 38, median = 9.92, iqr = 6.06, max = 14.9, min = 0.71) showing significantly increased VTCN1 expression in ACC compared to non-ACC samples and a nonsignificant trend towards increased VTCN1 expression in ACC1 (Wilcoxon test, two-sided). C: Exemplary staining of VTCN1 in an ACC sample reveals strong staining intensity on tumor cells. D: VTCN1 expression by RNAseq was significantly correlated with VTCN1 staining intensity by IHC (intensity * percentage positive cells), with the exception of 3 outliers (highlighted in red). The shaded area represents the 95% confidence interval of the linear model. E: Expression of VTCN1 in ACC malignant cells. UMAP shows non-integrated data. Cells are clustered by donor. F: UMAP of malignant cells in ACC colored by assigned cell type (myoepithelial- or luminal-like) revealed an association between VTCN1 expression and cell type. G: Violin plot with expression of VTCN1 in myoepithelial- vs. luminal-like malignant cells showed significantly higher expression in luminal ACC cells (luminal: n = 2615, median = 1.70, iqr = 0.88, max = 3.61, min = 0.38; myoepithelial: n = 1033, median = 1.20, iqr = 0.63, max = 3.05, min = 0.26). Cells expressing no VTCN1 were removed (Wilcoxon test, two-sided).