Characteristics of the oral and gastric microbiome in patients with early-stage intramucosal esophageal squamous cell carcinoma

Abstract

Background: Oral microbiome dysbacteriosis has been reported to be associated with the pathogenesis of advanced esophageal cancer. However, few studies investigated the potential role of oral and gastric microbiota in early-stage intramucosal esophageal squamous carcinoma (EIESC).

Method: A total of 104 samples were collected from 31 patients with EIESC and 21 healthy controls. The compositions of oral and gastric microbiota were analyzed using 16 S rRNA V3-V4 amplicon sequencing. Linear discriminant analysis effect size (LEfSe) analysis was performed to assess taxonomic differences between groups. The correlation between oral microbiota and clinicopathological factors was evaluated using Spearman correlation analysis. Additionally, co-occurrence networks were established and random forest models were utilized to identify significant microbial biomarkers for distinguishing between the EIESC and control groups.

Results: A total of 292 oral genera and 223 species were identified in both EIESC and healthy controls. Six oral genera were remarkably enriched in EIESC groups, including the genera Porphyromonas, Shigella, Subdoligranulum, Leptotrichia, Paludibacter, and Odoribacter. LEfSe analysis identified genera Porphyromonas and Leptotrichia with LDA scores > 3. In the random forest model, Porphyromonas endodontalis ranked the top microbial biomarker to differentiate EIESC from controls. The elimination rate of Porphyromonas endodontalis from the oral cavity to the stomach was also dramatically decreased in the EIESC group than controls. In the microbial co-occurrence network, Porphyromonas endodontalis was positively correlated with Prevotella tannerae and Prevotella intermedia and was negatively correlated with Veillonella dispar.

Conclusion: Our study potentially indicates that the dysbacteriosis of both the oral and gastric microbiome was associated with EIESC. Larger scale studies and experimental animal models are urgently needed to confirm the possible role of microbial dysbacteriosis in the pathogenesis of EIESC. (Chinese Clinical Trial Registry Center, ChiCTR2200063464, Registered 07 September 2022, https://www.chictr.org.cn/showproj.html?proj=178563).

Keywords: Amplicon sequencing analysis; Early esophageal cancer; Gastric microbiome; Intramucosal esophageal squamous carcinoma; Oral microbiome.

Fig. 1

The flowchart of the study

Fig. 2

Comparisons of microbial diversity. A, C, and E: the boxplot of alpha diversity between the EIESC and controls using the Chao1 (A), Shannon(C), and Simpson index (E). B, D and F: the Rarefaction curves of the diversity detected compared with the predicted diversity. The x-axis represents the number of sequences sampled while the y-axis represents the estimated Chao1 (B), Shannon index (D), and Simpson index (F). G: PCoAs of Bray‒Curtis distances on the microbiota distributions. Each dot represents a patient with EIESC or controls. Points clustered in red and blue represent the gastric microbial composition of the EIESC and controls, whereas the points clustered in yellow, blue represent the oral microbial composition of the EIESC and controls. EIESC: early-stage intramucosal esophageal squamous cell carcinoma; PCoAs: principal coordinate analyses. H: UpSet plot of differently-distributed taxa. The left graph represents the total number of differently-distributed species (X-axis) in EEC saliva (EEC_o), Control saliva (NC_o), EEC gastric biopsy (EEC_g), and control gastric biopsy groups (EEC_g) (Y-axis). The right graph represents the intersection of sets of species in multiple groups. Each column corresponds to a group or set of groups (dots connected by lines below the X-axis) containing the same species. The number of species in each set appears above the column, while groups shared are indicated in the graphic below the column. *, **, *** stands for p-value < 0.01, 0.005 and 0.001, respectively). EEC: early esophageal cancer

Fig. 3

The differential analysis of microbiota compositions between EIESC and controls at the phylum and genus level. A: the vertical bar chart presenting the oral microbiota compositions between EIESC and controls at the phylum level. The x-axis represents each sample and its group, and the y-axis represents the relative abundance. B-F: Boxplots showing the relative abundance of Firmicutes, Bacteroidetes, Proteobacteria, Firmicutes/Bacteroidetes (F/B) ratio, and Firmicutes/Proteobacteria (F/P) ratio in saliva samples. G: the vertical bar chart presenting the gastric microbiota compositions between EIESC and controls at the phylum level. H-L: Boxplots showing the relative abundance of Firmicutes, Bacteroidetes, Proteobacteria, F/B ratio, and F/P ratio in gastric biopsy samples. M-N: the vertical bar chart presenting the oral (M) and gastric (N) microbiota compositions between EIESC and controls at the phylum level. O: Volcano plot: the log2 fold-change indicates the mean relative abundance for each taxon. Each dot represents one genus. The blue dots represent no significant expression difference between the MHO and control groups, the red dots represent EIESC-enriched genus

Fig. 4

The differential analysis of microbiota compositions between EIESC and controls at the species level and microbial correlation with clinicopathological factors. A: Manhattan plots showing the distributions of each oral species identified in EIESC and individuals. Significantly-enriched species are depicted as transparent triangles, significantly-depleted species are presented as inverted solid triangles, and species with no statistical significance are depicted as full circles. The color of each dot represents the different phylum affiliations, and the size stands for their relative abundance. The light-green and light-blue boxes are used to denote different phylum groups. B: boxplots showing the abundance (Log 2 transformed) of typical genera in four different groups. The Wilcoxon Test was performed and *, **, *** stands for p-value < 0.01, 0.005 and 0.001, respectively). C: Visualization of the Mantel test. The triangle on the right side represents pairwise comparisons of clinically relevant factors with a color gradient denoting Spearman correlation coefficients. The potentially beneficial genus and potentially harmful genus were related to each clinical factor, respectively, using partial (geographic distance– corrected) Mantel tests. Edge width corresponds to the Mantel r statistic for the corresponding distance correlations, and edge color denotes the statistical p significance value. D: Heatmap matrix plot of Spearman’s correlation coefficients (ρ) among significantly enriched or delpeted species. The absolute value of ρ is indicated by a color code explained in the legend. The blue color indicates a positive correlation, whereas red represents a negative one. The scale of a square is proportional to ρ2. Cells above the matrix diagonal refer to specific ρ values and their statistical significance (p-value). Significance levels p < 0.05, p < 0.01, and p < 0.001 are indicated by *, **, and ***, respectively, whereas p > 0.05 is presented p explicitly

Fig. 5

Identification of the oral microbial biomarkers. A: LEfSe analysis. Plot of LDA Effect Size. The length of the bar column represents the LDA score. The figure shows the oral microbial taxa with significant differences between the EIESC (orange) and Control (green) (LDA score > 2). B: Random Forest model of the representative 30 microbial biomarkers to predict EIESC based on their mean decrease scores of the optimal model performance. C: boxplots showing the different abundance of Porphyromonas endodontalis in four different groups (Left boxplot), and the elimination rate of Porphyromonas endodontalis from the oral cavity to the stomach (Right boxplot). Significance levels p < 0.05, p < 0.01, and p < 0.001 are indicated by *, **, and ***, respectively, whereas p > 0.05 is presented p explicitly. D-F: Correlation plot of Porphyromonas endodontalis with strong correlation with three species, including Prevotella tannerae (D), Prevotella intermedia (E) and Veillonella_dispar (F)

Fig. 6

Co-occurrence network visualization of the oral (A) and gastric (B) microbial interactions in the EIESC individuals. The lines connecting nodes (edges) represent a positive (light green) or negative (red) co-occurrence relationship. The color of each dot represents the different taxonomic affiliations of the species (phylum level), the width of the edges reflects the absolute value of correlation coefficients, and the size corresponds to their relative abundance