Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: While the bacterial microbiome associated with oral cavity squamous cell carcinoma has been extensively investigated, the role of intratumoral bacteria in laryngeal squamous cell carcinoma (LSCC) remains less understood. This study seeks to explore the relationships between intratumoral bacteria in LSCC and clinicopathologic features.
Methods: DNA was extracted from 45 laryngeal tumors and 27 adjacent normal tissue samples from 18 patients with early-stage (stage 1-2) and 27 patients with advanced-stage (stage 3-4) LSCC treated between 2009 and 2020. Bacterial 16S rRNA gene (V4 region) sequencing (Zymo Inc.) was performed on formalin-fixed, paraffin-embedded (FFPE) samples. Due to the challenges posed by FFPE, including DNA fragmentation and degradation, we used optimized DNA isolation protocols, appropriate controls, and downstream computational pipelines for filtering bacterial contaminants. Microbial community composition was compared across clinical stages using α- and β-diversity metrics after rarefaction. Differential abundance analysis was conducted using ANCOMBC, adjusting for multiple testing with a false discovery rate (pFDR) threshold of 0.05. Functional profile predictions were predicted using PICRUSt2.
Results: In the advanced-stage group (N=27), the supraglottis was the most commonly affected subsite (66.7%), while the majority of early-stage tumors (N=18) were glottic (78%). The most abundant genera in the cohort were Streptococcus (11.95%), Corynebacterium (8.15%), Prevotella (6.57%), Lactobacillus (5.74%), Staphylococcus (4.32%), and Fusobacterium (4.13%). There were no significant differences in α-diversity between the tumor and adjacent normal tissues, but β-diversity was significantly different (Unifrac, p < 0.05). Among advanced-stage patients, those with lymph node metastasis showed increased α-diversity (chao1, p < 0.01) and β-diversity (Unifrac, p = 0.03) compared to those without metastasis. Fusobacterium was significantly more abundant in tumors with lymph node involvement (LogFC = 2.43, pFDR < 0.05), while Proteobacteria was decreased (LogFC = -1.68, pFDR < 0.05). Metagenomic functional predictions indicated enrichment of glycerolipid metabolism pathways in patients with lymph node metastasis (pFDR < 0.05).
Conclusions: This study provides a comprehensive profile of the intratumoral bacterial microbiome in LSCC, highlighting increased bacterial diversity in advanced-stage tumors with lymph node involvement. The elevated abundance of opportunistic pathogen Fusobacterium and decreased Proteobacteria, along with enriched glycerolipid metabolism, suggest that bacterial communities in the tumor microenvironment may play a role in LSCC metastasis. These findings could indicate that the unique bacterial communities found in the microenvironment of LSCC influence metastasis via bacterial metabolic products. Further mechanistic studies are needed to determine the potential role of pathogenic bacteria in promoting lymph node metastasis in patients with LSCC.