A large prospective study identified specific oral bacteria associated with an increased risk of developing head and neck squamous cell carcinoma.

In the study, published in JAMA Oncology, researchers analyzed oral wash samples from 236 patients who later developed head and neck squamous cell carcinoma (HNSCC) and 485 matched controls across three large U.S. cohorts. Using whole-genome sequencing and internal transcribed spacer sequencing, they characterized the oral bacterial and fungal microbiomes, respectively.

Oral bacterial microbiome analysis was conducted using whole-genome shotgun sequencing. Fungal microbiome analysis employed internal transcribed spacer sequencing. The researchers used Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) to assess the associations between microbial taxa and HNSCC risk.

Periodontal pathogen complexes were examined using logistic regression. A microbial risk score was constructed based on the relative abundance of risk-associated bacteria, with weights assigned according to effect sizes determined by ANCOM-BC.

The study included participants from the American Cancer Society Cancer Prevention Study II Nutrition Cohort (ACS-CPS-II); the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO); and the Southern Community Cohort Study (SCCS). HNSCC cases were identified during a mean follow-up of 5.1 years.

Participant characteristics included:

• Mean age of HNSCC cases: 60.9 years (standard deviation [SD] = 9.5)
• Sex distribution of cases: 24.6% female
• Race and ethnicity: 36% Black
• HNSCC sites: 26.3% oral cavity, 26.7% pharynx, and 47.0% larynx.

Among the key findings were:

• Thirteen bacterial species were differentially associated with HNSCC risk. Notably, Prevotella salivae, Streptococcus sanguinis, and Leptotrichia species (oral taxon 212) were associated with a lower risk of developing HNSCC, while several rare species were linked to an increased risk.
• The red and orange periodontal pathogen complexes were moderately associated with increased HNSCC risk.
• A microbial risk score summarizing 22 bacteria was associated with a 50% increase in HNSCC risk per 1-SD increase (odds ratio [OR] = 1.50, 95% confidence interval [CI] = 1.21–1.85).
• No significant associations were found between fungal species and HNSCC risk.

The study found no statistically significant differences in overall bacterial alpha or beta diversity between HNSCC cases and controls. However, 13 specific bacterial species were associated with HNSCC risk. Prevotella salivae, Streptococcus sanguinis, and Leptotrichia species were associated with lower HNSCC risk. Additionally, four species in Proteobacteria (Eikenella corrodens, Simonsiella muelleri, Rodentibacter pneumotropicus, and Pasteurella multocida) were also linked to lower risk.

The red and orange periodontal pathogen complexes were moderately associated with HNSCC risk (OR =1.06 per 1 SD, 95% CI = 1.00–1.12). Individual complex associations were:

• Red complex: OR = 1.07 (95% CI = 0.99–1.17)
• Orange complex: OR = 1.10 (95% CI = 1.00–1.21).

The microbial risk score, created based on 22 bacteria (13 identified by ANCOM-BC and 9 from the red/orange complexes), showed a significant association with HNSCC risk. This association remained consistent across disease sites (oral cavity, pharynx, and larynx) and was not substantially modified by tobacco use, alcohol consumption, HPV-16 status, or follow-up period.

Cohort-specific microbial risk score results (OR per 1-SD increase):

• ACS-CPS-II: 2.13 (95% CI = 1.29–3.52)
• PLCO: 1.23 (95% CI = 0.82–1.84)
• SCCS: 1.47 (95% CI = 1.11–1.96).

Fungal microbiome analysis revealed no significant associations between any fungal species, including Candida, and HNSCC risk. The most abundant fungal species identified were:

• Malassezia restricta (20% relative abundance)
• Candida albicans (4.9% relative abundance)
• Saccharomyces cerevisiae (2.7% relative abundance).

The study employed rigorous quality control measures, including blinded random repeated samples, positive microbial controls, and negative controls. Intraclass correlation coefficients for quality control samples ranged from 0.90 to 1 for whole-genome sequencing and 0.62 to 1 for internal transcribed spacer sequencing.

Limitations of the study included potential confounding by unmeasured factors, selection bias caused by loss to follow-up, and the observational nature of the study, which limited causal inference. Additionally, the use of oral wash samples may not have fully captured the complexity of oral biofilms.

One study investigator reported receipt of grants from the National Institutes of Health outside the submitted work. No other disclosures were reported.