The tumor microbiome in head and neck cancer: Insights into the complexity, distribution, and differential regulation of metabolic pathways between HPV-positive and HPV-negative subtypes

Recent studies have underscored the influence of intratumoral microbiota on cancer biology, including head and neck squamous cell carcinoma (HNSCC). However, the relationship between microbial distribution, survival outcomes, and tumor metabolism remains poorly understood. In this study, we investigated the presence, diversity, and clinical relevance of intratumoral microbiota in HNSCC using immunohistochemistry and 16S rDNA amplicon sequencing. Analyzing a tissue microarray of over 150 HNSCC samples, we detected both Gram-positive and Gram-negative bacterial components, with Gram-negative bacteria being more prevalent (44.1% vs. 18.9%). Substantial heterogeneity was observed both within tumors and between individuals. Microbial 16S rRNA was identified in 37.4% of primary HNSCC samples (37/99), with microbial load varying considerably across cases. Firmicutes was the most dominant phylum, followed by Bacteroidota and Proteobacteria . Microbial composition was significantly associated with HPV status, gender, age, and alcohol consumption. Importantly, we identified distinct metabolic pathways enriched in HPV+ and HPV− tumors. The methylaspartate cycle was upregulated in HPV+ cases, while L-glutamate degradation VIII was predominant in HPV− tumors. Despite these differences, the presence of intratumoral microbiota was not significantly associated with patient survival.. In conclusion, our findings confirm the presence of diverse bacterial communities within HNSCC tumors, shaped by clinical and demographic variables. While microbiota did not correlate with survival outcomes, their influence on tumor metabolism and microenvironment warrants further investigation. These insights lay the groundwork for exploring microbiome-targeted strategies as adjuncts to conventional cancer therapies. • Intratumoral microbiota are present in head and neck cancer • Abundance: Gram-negative bacteria > Gram-positive bacteria, heterogenic between tumors • Microbial composition is associated with demographic and clinical variables • Potential metabolic reprogramming in tumors is linked to HPV status • Intratumoral microbiota are not significantly associated with patient survival