Gut microbiota and metabolomic profiling in Relapsed/Refractory lymphoma

Abstract Gut Microbiota and Metabolomic Profiling in Relapsed/Refractory Lymphoma Objective: The gut microbiota, as a crucial component of the human body, plays a significant role in various diseases. This study investigates the differences in gut microbiota structure and metabolic products between patients with relapsed/refractory lymphoma and newly diagnosed lymphoma patients, aiming to identify therapeutic targets through metabolomics to advance treatment strategies for relapsed/refractory lymphoma. Methods: This study enrolled 14 patients with relapsed/refractory lymphoma and 7 newly diagnosed lymphoma patients admitted to Shanxi Bethune Hospital, Third Clinical Medical College of Shanxi Medical University, between November 2023 and December 2024. Fecal samples were collected from both groups of lymphoma patients and analyzed using 16S rDNA sequencing to assess alpha diversity, beta diversity, species composition, and significant differences. Untargeted metabolomics was employed to screen and perform enrichment analysis of differential metabolites between the two groups. Statistical methods included two-tailed paired t-tests, non-parametric Wilcoxon signed-rank tests, and R-based bioinformatics frameworks. Results: In terms of alpha diversity and beta diversity analyses, there were no significant differences between the two groups. Regarding species composition, the abundances of p-Actinobacteriota, p-Bacilli, c-Actinobacteriota, o-Lactobacillales, o-Bifidobacteriales, f-Enterobacteriaceae, f-Eubacterium, f-Bifidobacteriaceae, g-Escherichia-Shigella, and g-Enterococcus were increased in relapsed/refractory lymphoma. Conversely, p-Firmicutes, p-Bacteroidota, c-Clostridia, o-Oscillospirales, f-Lactobacillaceae, g-Faecalibacterium, and g-Klebsiella were reduced. In the analysis of significant species differences, c-Alphaproteobacteria was increased in relapsed/refractory lymphoma, while o-Erysipelotrichales, f-Morganellaceae, g-Faecalibacterium, g-Agathobacter, g-Klebsiella, and g-UCG-005 were decreased. From the metabolomics compositional analysis, 87 differentially expressed secondary metabolites were detected, with 47 upregulated and 40 downregulated. After normalization, these metabolites were visualized in volcano plots and heatmaps, revealing distinct metabolic profiles between the two groups. Significantly upregulated metabolites included 3-Amino-4-methylpentanoic Acid, 2-Hydroxybutyric Acid, UDP-N-acetylglucosamine, Pantothenic Acid, Isoleucine, Glycine, and Alanine. The KEGG enrichment analysis of differential metabolites highlighted significant enrichment in pathways such as Protein digestion and absorption, Mineral absorption, ABC transporters, Central carbon metabolism in cancer, Aminoacyl-tRNA biosynthesis, and 2-Oxocarboxylic acid metabolism. Conclusion： 1.In patients with relapsed/refractory lymphoma, butyrate-producing bacteria such as Faecalibacterium and Agathobacter are reduced in the gut, leading to diminished protection of the intestinal mucosa, which may contribute to the pathogenesis of relapsed/refractory lymphoma.The gut microenvironment of relapsed/refractory lymphoma patients exhibits enhanced central carbon metabolism in cancer and amino acid metabolism.The significant upregulation of UDP-N-acetylglucosamine, isoleucine, and glycine suggests their potential as novel therapeutic targets for relapsed/refractory lymphoma. Key words : Gut Microbiota;Metabolomics; Relapsed/Refractory Lymphoma