Longitudinal changes in the gut microbiota of Vietnamese patients with colorectal cancer undergoing surgery and chemotherapy

Figure 1 Microbial diversity of the gut microbiota in patients with colorectal cancer at diagnosis (T0), after surgery (T1), and after chemotherapy (T2). A: Boxplots showing the Pielou’s evenness, observed amplicon sequence variants, Faith’s phylogenetic diversity, and Shannon’s diversity alpha diversity indices. P values were calculated with the Friedman test, and the Nemenyi test for pairwise comparisons when the Friedman test was significant; B: Principal coordinates analysis based on the unweighted and weighted UniFrac distances. Differences in beta diversity between timepoints were assessed using permutational multivariate analysis of variance; C: Heatmap showing the presence (red) and absence (light yellow) of gut microbiota genera. Only classified genera with significant differences across the three timepoints (Benjamini-Hochberg-adjusted P value < 0.05, Cochran’s Q test) are displayed. Clustering of genera on the left side of the heatmap was performed using Euclidean distances and Ward’s hierarchical algorithm, resulting in three major clusters (a, b, and c) based on their presence across samples. Colored bars at the top of the heatmap represent tumor size, tumor, node and metastasis stage, location, therapy response, treatment regimen, carcinoembryonic antigen concentration, and timepoint.

Figure 2 Temporal gut microbiota changes at the genus level during the treatment period. Differentially abundant bacterial genera at post-treatment timepoints relative to T0 (diagnosis) were identified using LinDA, MaAsLin2, and ANCOM-BC2. Differences between T1 (after surgery) and T0 and between T2 (after chemotherapy) and T0 are shown as log₂ fold changes. Only classified genera with significant differences between timepoints are presented. ^aAdjusted P < 0.05; ^bAdjusted P < 0.01; ^cAdjusted P < 0.001.

Figure 3 Bacterial genera with significantly different abundance between patient subgroups stratified by treatment regimen after chemotherapy (T2). Only genera with an uncorrected P value < 0.05 (Mann-Whitney U test) are displayed.

Figure 4 Heatmap showing the Spearman’s rank correlations between amplicon sequence variant abundance and serum biochemical markers. Spearman’s ρ values were computed for each amplicon sequence variant (ASV) in relation to each biochemical marker. Multiple testing was adjusted using the Benjamini-Hochberg method, and only ASVs or markers with adjusted P values < 0.05 are displayed. Hierarchical clustering of ASVs was performed using the Euclidean distance metric. ^aAdjusted P < 0.05. ASVs were labeled with genus (g_), or family (f_), or order (o_) when the genus-level classification was unavailable. ASV: Amplicon sequence variant; GOT: Glutamic oxaloacetic transaminase; GPT: Glutamic pyruvic transaminase; CEA: Carcinoembryonic antigen.