The microbiome and metabolome during pelvic radiotherapy
80% patients develop gastrointestinal (GI) symptoms during pelvic radiotherapy and half develop chronic symptoms. The triggering event is known enabling the identification of pathophysiological changes. Studies suggest radiation alters diversity in the gut microbiome with differences in volatile organic compounds (VOCs) stratified by GI toxicity. Metabolomic and microbiomic profiling has the potential to advance understanding of disease-related pathways to predict, prevent and treat GI toxicity.   This observational cross-sectional feasibility study aimed to test the study process and generate data to power a definitive trial. Patients undergoing radiotherapy for cervical, endometrial or bladder cancer were recruited and underwent longitudinal faecal sampling [day 1 of radiotherapy (baseline), week 4 (radiotherapy fraction 20) and 6 months] with prospective assessment of GI toxicity using inflammatory bowel disease questionnaire bowel subset (IBDQB) score at each timepoint. Participants were split into 2 groups on the basis of IBDQB score at week 4. Exploratory analysis was performed to identify differences in metabolome (gas-chromatography-mass spectrometry) and microbiome (16s rRNA sequencing) to identify areas for further investigation.  227 patients were screened. 69 patients were approached (endometrial 30; cervix 31; bladder 8). Recruitment rates per tumour group were 30% (9) for endometrial cancer patients; 13% (4) for cervix cancer patients and 50% (4) for bladder cancer patients. Seventeen patients were recruited over 18 months (mean age 61.6 +/- 15.3 years; 14 female). There was one withdrawal. Recruitment was lower than anticipated due to non-patient factors (staffing, COVID-19 pandemic and competing studies) and patient factors (feeling too overwhelmed, having too much on and feeling too unwell). GI toxicity was at its worst at week 4, improving by month 6. The highest toxicity group at week 4 had IBDQ scores of 36-57, the lowest toxicity group had scores of 61-69.  Metabolome analysis showed lower heptanal and octanal in baseline stool samples in patients with higher GI toxicity at week 4; lower (methyltrisulfanyl)methane in week 4 stool samples in patients with higher GI toxicity at week 4; higher butanoic acid and benzaldehyde in month 6 stool samples in patients with higher GI toxicity at 6 months. Whole group microbiome analysis showed a trend towards decreased alpha diversity at 4 weeks; no differences in beta diversity at each timepoint; and a trend towards an increase in Lachnoclostridium and decrease in Ruminococcaceae Incertae sedis at week 4 (no difference between the GI toxicity groups). Microbiome analysis split by high and low GI toxicity showed lower alpha diversity for the high GI toxicity group at each of the timepoint; no significant difference in beta diversity between the GI toxicity groups; more genera differentially abundant between the GI toxicity groups at 4 weeks, than at baseline or 6 months.  Recruitment was lower than anticipated , but once recruited the attrition was low. Exploratory analysis suggests heptanal and octanal may have a role as a biomarker for GI toxicity, and lower alpha diversity may predict GI toxicity, with Lachnoclostridium and Ruminococcaceae Incertae sedis bacteria of interest.