C16orf74 is a novel prognostic biomarker and associates with immune infiltration in head and neck squamous cell carcinoma

Abstract

Head and neck squamous cell carcinoma (HNSC) is a prevalent and aggressive malignancy with poor prognosis, underscoring the need for novel biomarkers and therapeutic strategies. This study investigates the role of C16orf74 as a potential diagnostic and prognostic biomarker in HNSC. Bioinformatics analyses revealed that C16orf74 is significantly overexpressed in HNSC and is associated with advanced disease stages, therapy resistance, and shorter overall and progression-free survival. A prognostic nomogram integrating C16orf74 expression with clinicopathological features demonstrated robust predictive performance. Functional enrichment and immune infiltration analyses suggest that high C16orf74 expression might contribute to an immunosuppressive tumor microenvironment by reducing key immune cell populations, such as B cells, T cells, and natural killer cells, which are critical for anti-tumor immunity. Moreover, C16orf74 expression was inversely associated with immune checkpoint expression and immunotherapy response, highlighting its potential as a predictive biomarker for immune checkpoint blockade (ICB) efficacy. Drug sensitivity analyses identified potential therapeutic agents, including arsenic trioxide, carmustine, vincristine, quercetin, and carboplatin for patients with high C16orf74 expression. These findings highlight the potential of C16orf74 as a biomarker and therapeutic target to improve HNSC management.

Fig 1. Elevated expression of C16orf74 in HNSC and other cancers.

Pan-cancer analysis of C16orf74 expression in normal and tumor tissues from TCGA datasets (a). Paired analysis of C16orf74 expression in tumor and adjacent normal tissues from TCGA datasets (b). Comparative analysis of C16orf74 expression across multiple cancer types (c). Boxplot and paired plot of C16orf74 expression in HNSC tumor and normal tissues from TCGA datasets (d-e). ROC curve analysis of C16orf74 expression in HNSC (f). Independent validation of C16orf74 expression in HNSC using GSE23558, GSE30784, GSE31056, and GSE184616 datasets (g-j). TCGA, The Cancer Genome Atlas; HNSC, head and neck squamous cell carcinoma; ROC, receiver operating characteristic. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

Fig 2. C16orf74 is associated with adverse clinical characteristics of HNSC.

Association of C16orf74 expression with age (a), gender (b), primary therapy outcome (c), tumor status (d), clinical T stage (e), clinical N stage (f), clinical M stage (g), clinical stage (h), pathologic T stage (i), pathologic N stage (j), pathologic M stage (k), and pathologic stage (l) based on TCGA database analysis. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

Fig 3. Elevated C16orf74 expression is associated with poor prognosis in HNSC patients.

Pan-cancer univariate Cox regression analysis of C16orf74 expression from TCGA datasets (a). Kaplan-Meier survival analysis of OS (b) and PFS (c) in HNSC patients from TCGA datasets. Kaplan-Meier survival analysis of OS in HNSC patients from the GSE42743 dataset (d).

Fig 4. Gene mutation analysis of C16orf74 and its association with TMB in HNSC.

Alteration frequency of C16orf74 across various cancer types based on the cBioPortal database (a). Mutation profiles of patients with low (b) and high (c) C16orf74 expression levels in HNSC. Comparison of mutation frequencies for specific genes between low and high C16orf74 expression groups (d). Comparison of TMB between low and high C16orf74 expression groups in HNSC (e). Kaplan-Meier survival analysis of HNSC patients stratified by C16orf74 expression levels and TMB (f). HNSC, head and neck squamous cell carcinoma; TMB, tumor mutation burden. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5. Constructed nomogram based on C16orf74 predicts prognosis in HNSC patients.

Nomogram integrating C16orf74 expression and clinicopathological factors for predicting 1-, 3-, and 5-year OS in HNSC patients (a). Calibration curves of the nomogram for 1-, 3-, and 5-year OS (b). Time-dependent C-index comparison between the nomogram and other clinical indicators (c). Kaplan-Meier survival analysis of OS in HNSC patients stratified by nomogram scores (d). ROC curves showing the nomogram’s predictive performance for 1-, 3-, 5-year, 7-year and 9-year OS. (e). HNSC, head and neck squamous cell carcinoma; OS, overall survival; ROC, receiver operating characteristic.

Fig 6. WGCNA and functional enrichment analysis.

Clustering dendrogram of genes based on a topological overlap matrix (a). Module-trait relationships showing the correlation between gene modules and C16orf74 expression levels (b). Yellow module genes scatter plots (c). GO (d), KEGG (e) and hallmark pathway (f) enrichment analysis of yellow module genes. WGCNA, weighted gene co-expression network analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig 7. GSEA analysis.

Hallmark pathway enrichment analysis of C16orf74-associated signaling pathways in the TCGA dataset (a). GSEA enrichment plots for immune-related pathways, including allograft rejection (b), complement activation (c), IL2-STAT5 signaling (d), IL6-JAK-STAT3 signaling (e), inflammatory response (f), and interferon-gamma response (g). GSEA, gene set enrichment analysis; TCGA, The Cancer Genome Atlas.

Fig 8. C16orf74 is involved in immune cell infiltration in HNSC.

Comparison of ESTIMATE, immune, and stromal scores between low- and high-C16orf74 expression groups in HNSC patients (a). Heatmap of the infiltration of various immune cell types in low- and high-C16orf74 expression groups (b). Boxplots comparing infiltration levels of immune cell types between low- and high-C16orf74 expression groups (c). Correlation analysis between C16orf74 expression and immune cell infiltration (d). HNSC, head and neck squamous cell carcinoma.

Fig 9. Correlation and survival curves between C16orf74 expression and immune cell infiltration.

Immature B cells (a), activated B cells (b), effector memory CD4 T cells (c), eosinophils (d), natural killer cells (e), effector memory CD8 T cells (f), type 17 T helper cells (g), mast cells (h), and activated CD4 T cells (i).

Fig 10. Correlation analysis of C16orf74 expression with immunotherapy response.

Comparison of immune checkpoint expression levels between low- and high-C16orf74 expression groups in HNSC patients (a). Kaplan-Meier survival analysis between low- and high-C16orf74 expression groups in the IMvigor210 cohort (b). C16orf74 expression levels between patients with complete or partial response (CR/PR) and those with stable or progressive disease (SD/PD) in the IMvigor210 cohort (c). HNSC, head and neck squamous cell carcinoma; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease.

Fig 11. Correlation between C16orf74 expression and drug sensitivity.

Scatter plots (a) and boxplots (b) showing the negative correlation between C16orf74 expression and sensitivity to various drugs. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.