Association of Intratumoral Microbiota with Prognosis in Head and Neck Squamous Cell Carcinoma

PURPOSE: To evaluate whether intratumoral bacterial load and diversity are associated with survival outcomes in head and neck squamous cell carcinoma (HNSCC) and to examine their relationship with human papillomavirus (HPV) status. EXPERIMENTAL DESIGN: This retrospective cohort study included 312 adults with surgically treated, primary HNSCC at Montefiore Einstein Cancer Center (2000-2023). Intratumoral bacterial load was quantified via quantitative PCR (qPCR), and microbial diversity was assessed via 16S ribosomal RNA (rRNA) sequencing in 312 tumor and 34 paired normal tissues. HPV status was determined via p16 immunohistochemistry (IHC) and qPCR. Overall survival (OS)was the primary outcome. RESULTS: HNSCC tumors showed higher bacterial load and lower bacterial diversity compared with adjacent normal tissues. High bacterial load [hazard ratio (HR), 1.85; 95% confidence interval (CI), 1.31-2.61; P < 0.001] and low bacterial diversity (HR, 1.65; 95% CI, 1.19-2.28; P = 0.003) were independently associated with reduced OS, with the greatest risk in patients carrying both features (HR, 3; 95% CI, 1.76-5.09; P < 0.001). The high-risk bacterial features were less frequent in HPV-positive than in HPV-negative tumors (high load: OR, 0.46; 95% CI, 0.29-0.73; P = 0.001; low diversity: OR, 0.51; 95% CI, 0.32-0.81; P = 0.004), and their prognostic significance was more pronounced in HPV-negative cases. Taxonomic profiling revealed marked depletion of predominant bacterial taxa in HNSCC, especially in HPV-negative tumors. Notably, loss of the class TM7-3 and the orders Actinomycetales and Burkholderiales was specifically associated with poor HNSCC survival, including early mortality. CONCLUSIONS: High intratumoral bacterial load and low diversity are prognostic factors associated with survival in HNSCC, particularly in HPV-negative patients. Incorporating microbiome assessment into risk stratification may enhance prognostic precision and inform microbiota-directed therapeutic approaches.