Dirk S. Hoening

Protein network analysis of core-enriched genes within the hypoxia signature and TGF-β signaling from GSEA analysis. Clustering was performed using the STRING database. Three main clusters are depicted: hypoxia (blue color), TGF-β (green color), and EGFR (red color).

Molecular classification and validation of hypoxia-immune signature in HNSCC. A, Heatmap of molecular subgroups in the Moffitt (n = 228) HNSCC cohort. RNA expression data were z-normalized, each row represents a single gene in the hypoxia-immune signature gene list, each column represents a patient sample. Samples were reordered according to its molecular subgroup: Immune (blue), Mixture (black), and Hypoxia (red). B, Kaplan–Meier survival plots for overall survival stratified according to the Hypoxia-Immune signature for the Moffitt (n = 184) HNSCC patient cohort.

Hypoxia subgroup has immunosuppressive TME. Boxplots showing cell counts distribution of different immune markers CAIX+ cells representing hypoxia marker (A), CD3+ T cells (B), CD8+ cytotoxic T cells (C), PCK+ tumor cells expressing PD-L1 (D), HLA-DR+ APCs (E), and CD8+ T cells/FOXP3+ Tregs (F), among the three subgroups in the TMA mIHC cohort (n = 90). Statistical analysis was performed by ANOVA test and followed by Tukey post hoc tests. G, Representative mIHC images showing that the average numbers of immune cells expressing CD3+ T cells (green color), CD8+ cytotoxic T cells (yellow color), CD69+ activated T cells (red color), CD103+ resident memory T cells (cyan color) within 20 μm of a PCK+ cell (orange color) was statistically significantly lowest in the Hypoxia subgroup compared with the Mixture and Immune subgroups. Nuclei are shown in blue. Individual TMA core was considered as a region of interest (ROI). Bottom panel represents the zoom-in distance proximity and spatial distribution between a tumor cell and an immune cell within the TME.

(A, B) Heatmap of molecular subgroups in two different HNSCC datasets (A) TCGA and (B) CPTAC. RNA expression data was z-normalized, each row represents a single gene in the hypoxia-immune signature gene list, each column represents a patient sample. Samples were reordered according to its molecular subgroup: Immune (blue), Mixture (black) and Hypoxia (red). (C) Kaplan–Meier survival plots for overall survival stratified according to the hypoxia-immune signature for TCGA (n = 518) HNSCC patient cohorts.

Supplementary Table S6. Core enriched genes within the Hypoxia signature

Disease control given pembrolizumab or nivolumab monotherapy in Moffitt retrospective study cohort

Hypoxia subgroup has an enrichment of EGFR pathway. Pearson correlation coefficient for the TIMEx scores for the EGFR pathway and the Hypoxia gene signature among the TCGA (A), CPTAC (B), and Moffitt (C) HNSCC datasets. Expression distribution of EGFR RNA (D), protein (E), and phosphoprotein (F) levels among the three subgroups in the CPTAC HNSCC cohort.

Supplementary Table S2. CPTAC Cohort

GSEA of hallmark pathways in TCGA and Moffitt cohorts. TCGA (A–C) and Moffitt head and neck squamous cell carcinoma (D–F) cohorts. The gene expressions were compared for pathways enriched in: (A, D) Immune versus Hypoxia. Red: enriched in Immune, Blue: enriched in Hypoxia. B and E, Immune versus Mixture. Red: enriched in Immune, Blue: enriched in Mixture. C and F, Hypoxia versus Mixture. Red: enriched in Hypoxia, Blue: enriched in Mixture. Indicated are the top 10 most differentially expressed pathways in Cancer Hallmarks gene sets. NES: Normalized enrichment score.

Head and neck squamous cell carcinoma (HNSCC) has one of the most hypoxic and immunosuppressive tumor microenvironments (TME) among solid tumors. However, there is no proven therapeutic strategy to remodel the TME to be less hypoxic and proinflammatory. In this study, we classified tumors according to a Hypoxia-Immune signature, characterized the immune cells in each subgroup, and analyzed the signaling pathways to identify a potential therapeutic target that can remodel the TME. We confirmed that hypoxic tumors had significantly higher numbers of immunosuppressive cells, as evidenced by a lower ratio of CD8+ T cells to FOXP3+ regulatory T cells, compared with nonhypoxic tumors. Patients with hypoxic tumors had worse outcomes after treatment with pembrolizumab or nivolumab, anti-programmed cell death-1 inhibitors. Our expression analysis also indicated that hypoxic tumors predominantly increased the expression of the EGFR and TGFβ pathway genes. Cetuximab, an anti-EGFR inhibitor, decreased the expression of hypoxia signature genes, suggesting that it may alleviate the effects of hypoxia and remodel the TME to become more proinflammatory. Our study provides a rationale for treatment strategies combining EGFR-targeted agents and immunotherapy in the management of hypoxic HNSCC.Significance:While the hypoxic and immunosuppressive TME of HNSCC has been well described, comprehensive evaluation of the immune cell components and signaling pathways contributing to immunotherapy resistance has been poorly characterized. We further identified additional molecular determinants and potential therapeutic targets of the hypoxic TME to fully leverage currently available targeted therapies that can be administered with immunotherapy.