Fig. 1

A Workflow of the main analyses. B Forest plot representing the association of average expression of 125 genes included in the 26 immune gene sets and the clinical variables in 520 HNSCC patients from TCGA. Results of the linear regressions are shown as Odds Ratio with confidence intervals at 95%. C-D Kaplan–Meier curves of HNSCC patients from TCGA cohort with high or low Immune Scores evaluated for overall survival and progression free survival (panels C and D, respectively). Differences between curves were evaluated by logrank test. Hazard ratios with 95% confidence intervals were assessed by Cox Hazard regression models. Immune Scores were evaluated as the positive and negative z-scores of the average expression of the 125 genes composing the immune gene sets. E Overall Survival in a cohort of 108 HPV-negative HNSCC patients (Huang et al.). Patients were divided based on high and low levels of the Immune Score. Differences between curves were evaluated by log-rank test. F Distributions of the gene signature composed by the average expression of 125 genes of the immune gene sets by TP53 mutation and TP53 mutation carried on other mutations among FAT1, CDKN2A and PIK3CA in HNSCC patients (106 WT, 171 TP53 and 189 TP53 + mutX). P-values were evaluated by KruskalWallis test. G Distributions of the PDL1 protein among different mutational status subgroups from a set of 339 HNSCC patients evaluated by reverse phase protein array (RPPA) in the TCGA cohort. H Gene set enrichment analysis of co-mutated patients versus TP53 mutated patients in the TCGA HNSCC cohort. The size of the circles indicates the percentage of genes included in the pathway. Pathways are sorted by False Discovery Rate and normalized enrichment score (NES). The PI3K pathway and the MYC pathway activity were highlighted. For the analysis, we used the GSEA 4.2 software (https://www.gsea-msigdb.org/gsea/index.jsp) run in pre-ranked mode with HALLMARK pathways. I Pearson’s correlation between the mean expression of 26 immune gene sets (upper panel) and PD-L1 expression values (bottom panel) with the levels of expression of a 22 genes signature MYC dependent (Ganci et al.) in HNSCC patients from TCGA. A Multivariate regression models were built to adjust the differences of the genes between patients with high and low MYC signature. The models include T status, TP53 mutation, gender, smoking status and, HPV status. High and low expression of the MYC signature were evaluated by positive and negative z-scores of the mean gene expression, respectively. J qRT-PCR analysis of PD-L1 in Cal27, FaDu and Detroit 562 cell lines. Statistics (t-test): * p < 0.01, ** p < 0.005. K Flow cytometry analysis of PD-L1 surface expression in cell lines. Representative cell lines (color-coded) were harvested from their cultures and stained with CD274-PE mAb or control Ig for 30 min at 4 °C. Surface expression was assessed on single, live cells on the Attune NxT cytometer. Mean fluorescence intensity is shown. The staggered plot depicts cell line expression according their mutational status