Characterisation of gut microbiota in Malaysian cancer patients using V3-V4 region of 16S rRNA gene sequencing

Abstract

Recent studies suggested a potential connection between gut microbiota changes and cancer onset. However, conflicting results make it challenging to understand the role of gut microbiota dysbiosis in cancer, particularly in underrepresented populations like those in Southeast Asia. To address this gap, we analysed the diversity and composition of gut microbiota in 65 faecal samples, which included 48 from cancer patients with various malignancies and 17 from healthy controls. Patients were categorised into four groups: symptomatic patients undergoing cancer treatment, asymptomatic pre-treatment and during cancer treatment, and healthy controls. Genomic DNA was extracted, and the V3-V4 region of the 16 S rRNA gene was sequenced. Our findings revealed significant differences in the alpha diversity (p ≤ 0.05) between cancer patients and controls. Asymptomatic patients under treatment showed slightly lower alpha diversity than pre-treatment patients, but this difference was not statistically significant (p = 0.06). We identified 13 genera with over 20% difference in abundance between patient groups and controls. Asymptomatic patients receiving treatment and pre-treatment patients exhibited enrichment in Enterococcus, whereas Prevotella, Faecalibacterium, Brevundimonas, and Pseudomonas were significantly reduced compared to controls. Symptomatic patients had higher levels of Enterococcus and Staphylococcus, while Ruminococcus was enriched in asymptomatic patients. These underscore the distinct differences in gut microbiota composition between cancer patients and healthy individuals, particularly in symptomatic cases with potential biomarkers such as Enterococcus, Prevotella, and Faecalibacterium. Our study suggests that cancer treatment may not significantly alter the gut profile of cancer patients. Further research is needed to comprehend the implications of these findings fully.

Keywords: 16S rRNA sequencing; Cancer; Gut microbiota; Hypervariable V3-V4; Malaysia; Symptomatic and asymptomatic cancer patients.

Fig. 1

Dendrogram shows sample clustering based on taxonomic profiles with slight clustering of samples measured based on the types of cancer. ALL = acute lymphoblastic leukaemia; AML = acute myeloid leukaemia; JMML = juvenile myelomonocytic leukaemia; LCH = Langerhans cell histiocytosis.

Fig. 2

Alpha and beta diversity measured on cancer patients at pre-treatment (P) and treatment (T) adjusted for GI symptoms against healthy control. (a) Observed species richness at minimum sample depth of 4273, (b) Shannon effective index and (c) Shannon Effective Number. (d) PCoA plot based on the Bray-Curtis dissimilarity showed distinct clustering between cancer patients and controls. *Boxplot depicts the median, interquartile and extremities of the values. p-value calculated using Kruskal-Wallis t-test with *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Fig. 3

Taxonomic analysis of gut microbiota of cancer patients. (a) Taxonomic bar plot showing comparison of microbial composition at phylum level in symptomatic patients who are receiving treatment (S + T), asymptomatic and receiving treatment (A + T), asymptomatic and pre-treatment (A + P) and control. (b). Top 5 phyla with altered relative abundance in cancer patients at pre-treatment (P) and treatment (T) adjusted by GI symptoms. Pairwise comparison between two groups was marked as asterisk for significant differences in relative abundance based on Wilcoxon rank sum test (*p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). (c) Bacillota/Bacteroidota (B/B) ratio in abundance of cancer patients and control.

Fig. 4

Heatmap of differentially abundant genus between S + T, A + T, A + P and control with more than 20% abundance (Kruskal-Wallis test, p ≤ 0.05).

Fig. 5

Genera with significant abundance between cancer patients at pre-treatment (P) and treatment (T) adjusted by GI symptoms and control. Genera with altered relative abundance in cancer patients by pairwise comparison between two groups was marked as asterisk for significant differences based on Wilcoxon rank sum test (*p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Fig. 6

Different abundances of microbial communities between cancer patients and healthy control. (a) Linear discriminative analysis (LDA) effect size (LEfSe) analysis between asymptomatic cancer patients (red) and healthy controls (green). (b) A cladogram displays the connection between the significantly different taxa at different levels with the clade as a branch of organisms that share a common ancestor.