Cross-comparison of microbiota in the oropharynx, hypopharyngeal squamous cell carcinoma and their adjacent tissues through quantitative microbiome profiling

Abstract

Aims: To clarify the absolute abundance of microbial communities on hypopharyngeal squamous cell carcinoma and their correlation to those in the oropharynx.

Methods: Clinical data, swabs, and tissue samples from 27 HPSCC patients were collected in this study and divided into three sampling groups: 19 oropharyngeal mucosa (OPM), 27 hypopharyngeal carcinomas tissues (HC), and 26 corresponding adjacent tissues (AT). Relative microbiome profiling (RMP), and quantitative microbiome profiling (QMP) of 16S rRNA amplicon sequencing were used for analysis.

Results: Beta-diversity showed that abundance and phylogenetic tree in OPM group were less when compared to either HC and AT. Although HC and AT were found to have similar microbiota, Bray-Curtis based beta-diversity still highlighted differences. Fusobacterium, Porphyromonas, Haemophilus, and Peptostreptococcus at the genus level in OPM were positively correlated with HC. After categorizing HC through TNM staging, the abundance of genera Fusobacterium, Parvimonas, and Dialister were found to be enhanced in higher T classifications (T3-4) and advanced stages (Ⅳ).

Conclusions: QMP yielded more comprehensive results than RMP. Dysbiosis was found in OPM groups and could be used to narrow down differential microbiome for the HC group. Genera of Parvimonas, Fusobacterium, and Dialister were deemed asrisk factors of advanced HPSCC.

Keywords: ASV; HPSCC; QIIME2; Quantitative microbiome profiling; cross-sectional study.

Figure 1.

The relative and absolute composition of OPM, HC, and AT in barplot and pie plot. (A) phylum level in barplot. (B) genus level in barplot. The amount of ASVs in these three groups presents a significant difference under Kruskal-Wallis test (HC-AT: p = 0.0002; HC-OPM: p = 0.0026; AT-OPM: p < 0.0001). Pie plot depicted the different proportions of genera in RMP (C-E) and QMP (F-H).

Figure 2.

The beta-diversity of HC, AT, and OPM groups analyzed using different methods. (A) Bray-Curtis method. (B) Jaccard method. (C) Unweighted UniFrac. The variation in these three groups showed the same tendency in both QMP and RMP. (D) In Weighted Unifrac, QMP showed no difference in microbiota variation in these three groups (p = 0.1475).

Figure 3.

Relative versus absolute abundance of microbiota network reconstruction. A total of 25 common microbiomes in HC group were cross-validated using QMP (upper triangle) and RMP (lower triangle). The taxa are ordered by the significance of the correlation between their QMP abundance and DNA count. The correlation coefficient was measured by Spearman’s ρ analysis and presented using different colors and sizes of circles. The color gradients on the matrix axes (blue: positive correlation; red: negative correlation) are shown.

Figure 4.

Bray-Curtis and Weighted UniFrac based beta-diversity in HC and AT groups. (A-B) Bray-Curtis based PCoA and NMDS presented significant differences in QMP (Adonis: p = 0.0005). However, the absolute abundance of Weighted UniFrac based PCoA and NMDS (C-D) and relative abundance of both Bray-Curtis and Weighted UniFrac based beta-diversity (E-H) showed no significant difference (Adonis: p > 0.05).

Figure 5.

The absolute abundance of microbiota through LefSe analysis in HC and AT groups. (A) A cladogram represents the microbiota in HC and AT. The brightness of each dot was proportional to its effect size. (B) Taxa were enriched in HC group (Red), and AT groups (Green), indicating the variation of microbial communities under LDA scores (LDA = 2), respectively.

Figure 6.

The absolute beta diversity and LEfSe analysis in HC and OPM groups. (A-B) Both PCoA and NMDS methods under Bray-Curtis analyses showed a significant difference in HC and OPM groups (Adonis: p = 0.0001). (C-D) Both PCoA and NMDS methods under Weighted UniFrac analyses showed significant differences in HC and OPM groups (Adonis: p = 0.0008). (E) A cladogram, on the left side, represents the OPM microbiota in HC and OPM. The brightness of each dot was proportional to its effect size. (F) Taxa were enriched in HC group (Red), and OPM groups (Green), indicating the variation of microbial communities under LDA scores (LDA = 2), respectively.

Figure 7.

Heatmap indicates the correlation of HC and OPM in QMP. The top 15 of high absolute abundance of genera in HC were screened out and had their correlations compared with OPM through Spearman’s correlation analysis. * p < 0.05; ** p < 0.01.

Figure 8.

Potential genuses of microbiota found in HC group under different T classification and TNM stagings. (A-F) The genera Prevotella (p = 0.0001), Solobacterium (p = 0.0008), Fusobacterium (p = 0.0024), Parvimonas (p = 0.0024), Dialister (p = 0.016) increased whereas the genus Pseudomonas (p = 0.009) was downregulated in advanced T classification (T3-T4), when compared to early T classification (T1-T2). (G-L) The genera Treponema (p = 0.0043), Parvimonas (p = 0.0043), Dialister (p = 0.0085), Fusobacterium (p = 0.0155), Solobacterium (p = 0.0366), Slackia (p = 0.0412) were higher in advanced TNM staging (IV), when compared to early TNM staging (II–III).