Gut microbial dysbiosis may predict diarrhea and fatigue in patients undergoing pelvic cancer radiotherapy: a pilot study

Abstract

Fatigue and diarrhea are the most frequent adverse effects of pelvic radiotherapy, while their etiologies are largely unknown. The aim of this study is to investigate the correlations between fatigue, diarrhea, and alterations in gut microbiota induced by pelvic radiotherapy. During the 5-week treatment of pelvic radiotherapy in 11 cancer patients, the general fatigue score significantly increased and was more prominent in the patients with diarrhea. The fatigue score was closely correlated with the decrease of serum citrulline (an indicator of the functional enterocyte mass) and the increases of systemic inflammatory proteins, including haptoglobin, orosomuoid, α1-antitrypsin and TNF-α. Serum level of lipopolysaccharide (LPS) was also elevated, especially in the patients with diarrhea indicating epithelial barrier breach and endotoxemia. Pyrosequencing analysis of 16S rRNA gene revealed that microbial diversity, richness, and the Firmicutes/Bacteroidetes ratio were significantly altered prior to radiotherapy in patients who later developed diarrhea. Pelvic radiotherapy induced further changes in fecal microbial ecology, some of which were specific to the patients with or without diarrhea. Our results indicate that gut microbial dysbiosis prior to radiation therapy may be exploited to predict development of diarrhea and to guide preventive treatment options. Radiation-induced dysbiosis may contribute to pelvic radiation disease, including mucositis, diarrhea, systemic inflammatory response, and pelvic radiotherapy-associated fatigue in cancer patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. General fatigue scores in different patient groups at indicated time points.

Each symbol represents a sample. Horizontal bar lines refer to mean value in each group. * indicates statistical differences between pre-treatment time point and 3rd or 5th week, respectively, in patients who developed diarrhea (p < 0.01, n = 6; paired t-test).

Fig 2. Biochemical systemic markers of enterocyte mass and inflammation in different patient groups at indicated time points.

Serum citruline, haptoglobin, orosomucid, and α1-antitrypsin were analyzed by ELISA, as described in Materials and Methods section. Differences within groups were analyzed by ANOVA followed Tukey post-hoc test. Differences at respective time points between the two groups were analyzed with were analyzed with unpaired t-test, *p<0.05, #p<0.01.

Fig 3. Serum concentrations of TNFα (A) and LPS (B) in patients receiving pelvic radiotherapy.

TNF and LPS were analyzed by ELISA, as described in Materials and Methods section. *, # indicates statistical differences between “No diarrhea” and “Diarrhea” groups at the respective time point (*p <0.05, #p<0.01, non-parametric paired t-test, n = 5–6).

Fig 4. Fecal microbial ecology is altered in cancer patients prior to radiotherapy.

Differences in microbial alpha diversity between healthy controls and cancer patients prior to radiotherapy who did or did not develop diarrhea is indicated by Shannon’s diversity index (A) and Chao1 species richness (B). (C) Firmicutes/Bacteroides ratio in the same groups of patients. * and # indicate statistically significant differences at p<0.05 and 0.01, respectively (ANOVA followed by Tukey post-hoc test).

Fig 5. Taxonomic analysis (relative abundance) of fecal microbiota at phylum and genus levels.

(A) relative abundance of five main bacterial phyla as well as unclassified “Others”, which include Chloroflexi, Deferribacteres, Chlorobi, Acidobacteria, Deinococcus-Thermus, Planctomycetes, Lentisphaerae, Spirochaetes, Synergistetes, Tenericutes, Verrucomicrobia and Cyanobacteria. (B) Selected genera statistically different between cancer patients prior to radiotherapy and healthy controls. * and # indicate significant differenence from healthy controls (unpaired Mann-Whitney test). Symbol of + indicates statistical differences between cancer patients who did or did not develop diarrhea as a result of radiotherapy (unpaired Mann-Whitney test). (C, D) The effects of radiotherapy on relative microbial abundance at the genus level between patients prior and after radiotherapy within “diarrhea” and “no diarrhea” groups (paired Mann-Whitney test). (E) Taxonomic differences at the genus level between cancer patients without and with diarrhea after radiotherapy (unpaired Mann-Whitney test). *p <0.05, #p <0.01, +p<0.01.