This study is a single-center, randomized, parallel controlled clinical trial conducted at the Affiliated Hospital of Jiangnan University in Wuxi, China. Participants with LSCRC and RSCC will receive prebiotic or placebo intervention during postoperative chemotherapy for three months. The study flow is shown in Fig. 1. Study procedures and data collection are shown in Fig. 2 based on SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) .
- Patients diagnosed with primary CRC who will be treated with XELOX chemotherapy;
- Patients aged 18–80 years;
- Patients with an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1, absolute number of neutrophils ≥ 1.5×109/L, platelets ≥ 100× 109/L, serum creatinine ≤1.5 times upper limit of normal (ULN), total bilirubin ≤1.5 times ULN, aspartate transaminase/alaninetransaminase≤2.5 times ULN and carcinoembryonic antigen within the normal range;
- Patients have not received preoperative neoadjuvant radiotherapy and chemical treatment;
- Written informed consent.
- Suffer from other tumors at the same time;
- Patient has other digestive tract diseases except gastrointestinal tumors, such as inflammatory bowel disease, acute gastroenteritis, etc.;
- Those who have taken antibiotics, drugs or food containing probiotics within 2 weeks;
- Those with active infections or those with mental illness, cardiovascular and cerebrovascular diseases, severe cardiopulmonary dysfunction, and other serious diseases that are not suitable for chemotherapy;
- Those who have undergone multiple courses of chemotherapy, extensive radiotherapy, advanced age, bone marrow metastasis, severe infection, adrenal insufficiency, and severe illness;
- Women during pregnancy;
- Patients who cannot receive treatment on time and cannot cooperate fully.
Patients who change medication regimen in the chemotherapy cycle, patients who receive antibiotics or other probiotics/ prebiotics during the intervention, individuals who do not take the prebiotics consistently, and individuals who suffer complications that may be associated with prebiotics will be excluded.
Randomization and blinding
After signing the informed consent form and complete baseline questionnaires, patients with LSCRC (n= 50) and RSCC (n= 50) will be randomly divided into prebiotic group (n=25) and control group (n=25) in a 1:1 allocation ratio, respectively. The random number will be generated by a computer and sealed in envelopes by an investigator who will not be involved in running the study, and placed in a safe place. Once a participant meets the enrollment conditions and signs the informed consent can open one envelope in the order of enrollment time. Prebiotic and placebo will be packaged in opaque bags without any graphics, and their appearance and smell are the same. A third party who has no direct involvement will stick the "A" and "B" codes on the packaging boxes. Before data analysis, participants and investigators will not know the contents of the bags, allocation, and study treatments.
Setting and interventions
Posters and leaflets will be accessible in the outpatient and inpatient departments of the hospital so that interested candidates with CRC can get in touch with investigators to participate in the screening process. The eligible participants will be invited for the first visit, and two trained investigators will explain the study procedures in detail to them: (1) The purpose of this study, whether there are risks and discomforts, whether it needs to be paid, whether it is completely voluntary, etc.; (2) The types of samples collected in this study and the details of the collection; (3) The personal information of the subjects, research-related measurement indicators, and physical examination information will be kept confidential. Informed consent will be voluntarily signed by individuals who agree to participate in this study. Basic information questionnaires, including body mass index, age, gender, and dietary habit etc., will be conducted through interviews. Blood and stool samples will be obtained as baseline samples from the participants before preoperative bowel preparation. During surgery, three samples from three different sites including tumor tissue, paracancerous tissue, and normal tissue will be collected in sterile tubes and stored in liquid nitrogen, then will be transferred to the -80℃ refrigerator until use. The participants will visit the hospital for postoperative chemotherapy about 3 weeks after surgery. Since the chemotherapy regimen will change with the cancer stage, only participants who receive XELOX chemotherapy (oxaliplatin + capecitabine) will continue to participate in this trial.
Participants in the prebiotic group (n = 25 per group) will be asked to consume prebiotic (XOS, 3 g/day) along with routine capecitabine therapy. Participants in the control group (n = 50 per group) will be asked to consume placebo (maltodextrin, 3 g/day). Prebiotics and placebo are white powder, packed in 3g/ bag. Since maltodextrin is colorless and tasteless after dissolution, it can reduce the chance of the placebo effect interfering with results. Each prebiotic bag (produced by Heagreen Company, Henan, China) contains 40.64% xylobiose, 27.75% xylotriose, 14.16% xylotetraose, 7.14% xylopentaose, 7.8% xylohexaose. Each box contains 30 bags. Participants will be instructed to mix one bag prebiotic or placebo with water and consume it every day. Participants will be given a box of prebiotics each time they visit the hospital for chemotherapy. The intervention will last for 12 weeks. Participants will be monitored for prebiotic consumption at each delivery of prebiotics and weekly telephone follow-up. Throughout the intervention, volunteers will be required to maintain their lifestyle, diet, as well as medications. They will be asked to collect stool and blood samples before (3 week) and after (15 week) the intervention and complete questionnaires in each chemotherapy cycle. Participants are asked to inform the investigators in time if there is any adverse event that may be associated with prebiotic. These adverse events will be recorded by investigators. Protocol modifications will be communicated to the ethics committee through an official modification request, and the participants will be notified by telephone.
Data collection and sample handling
The overall study design is depicted in Fig. 1. Assessment of participants will be conducted at the perioperative (baseline = week 0), pre-treatment (week 3), post-treatment (week 15), and in each chemotherapy cycle. The primary outcomes will be the differences in the microbiota composition at different CRC tumor sites and differences in gut microbiota composition, adverse reactions, blood concentration in patients with CRC treated for 12 weeks with XOS or placebo. The secondary outcomes will include other blood indicators, SCFAs concentration, QoL, and mental health.
Gut microbial composition
Tissue samples including tumor, para-carcinoma mucosa (2 cm away from the tumor), and normal mucosa (as far as possible from the tumor) will be obtained from LSCRC and RSCC participants during surgery. Each tissue will be cut into small pieces with a volume of 1 cubic centimeter and put into the cryopreserved tubes. After being frozen in liquid nitrogen, the tissue will be stored in the -80 ℃ refrigerator for later use.
A total of 6 g stool samples from each participant will be collected in the morning for intestinal flora examination at baseline prior to preoperative bowel preparation and at 3 weeks (pre-treatment) and 15 weeks (post-treatment). Briefly, participants will be instructed to: (1) exhaust urine before placing disposable stool tray to prevent contamination of stool; (2) pass stool on the tray; (3) use a sterile spoon to dig up about 2g stool from middle and posterior segment of stool and inserted into a sterilized screw cap containers marked with the participant's code and sampling date. For the purpose of reducing the change of microbiota composition in the stools, samples will be temporarily stored in a foam box with ice packs and transferred to the -80℃ refrigerator in the laboratory within two hours.
The total DNA of frozen samples will be extracted via QIAamp (QIAGEN) stool DNA kit according to manufacturer’s instructions. DNA integrity and size will be assessed by the NanoDrop ND-1000 spectrophotometer (Thermo, USA) and 1% agarose gel electrophoresis. DNA samples will be kept at -80℃ for later use. The bacterial 16S rRNA V3–V4 regions will be amplified by polymerase chain reaction (PCR). High-throughput sequencing will be performed on an Illumina platform.
A sequence similarity threshold of 97% will be used to select operational taxonomic units (OTUs) and then perform taxonomy assignments via the Usearch platform (version 7.1 http://drive5.com/uparse/). Principal co-ordinates analysis (PCoA) on weighted UniFrac distances will be measured on all OTUs by QIIME. Alpha diversity will be calculated through the Shannon index, Simpson index, and Chao1 metrics using mothur (version v.1.30.1 http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity). Linear discriminant analysis (LDA) will be performed on the analysis software LEfSe (http://huttenhower.sph.harvard.edu/galaxy/). Subsequently, the communities or species that have significant differences in the sample division will be selected.
Stool SCFA profiling
Stool samples aliquots of 50 mg g of dry weight each, will add with 50 μL phosphoric acid, 100 μL isohexanoic acid solution, and 400 μL diethyl ether to homogenize. After centrifugation, the supernatant will be separated for gas chromatography-mass spectrometry (GC-MS) analysis. Stool SCFA profiling will be conducted using an Agilent Technology 6890 GC with autosamplers and 5973 MS Detection and Chemstation Data System (Agilent, Singapore). Chromatographic conditions will be as follows: Agilent HP-INNOWAX capillary column (30 m *0.25 mm ID*0.25 μm); shunt injection, injection volume 1μL, shunt ratio 10:1; injection port temperature of 250℃; ion source temperature of 230℃; initial oven temperature of 90˚C, 10˚C/min up to 120˚C, 5˚C/min up to 150˚C, 25˚C/min up to 250˚C, and keep at 250˚C for 2 min. Helium will be used as gas carrier at a constant flow rate of 1 mL/min.
Venous blood samples will be collected before preoperative bowel preparation, at 3 weeks (pre-treatment) and 15 weeks (post-treatment). After fasting for 12 hours, venous blood will be collected into a coagulating tube in the morning and centrifuged at 3000 rpm for 10 minutes for obtaining the upper serum (Thermo, USA). In addition to some of the usual blood biochemical indexes, we will use commercial enzyme-linked immunosorbent assay (ELISA) kits to assess the levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon gamma (IFN-γ). Cellular immune indices, including T-lymphocytes (CD3+), helper inducer T cells (CD3+CD4+), suppressor/cytotoxic T cells (CD3+CD8+) will be measured by BD-FACS Canto II flow cytometer (Becton Dickinson, USA).
For the purpose of evaluating the bioavailability of chemotherapy drugs in participants, the blood concentration of capecitabine will be measured at 15 weeks (after intervention). On the third day of the fifth cycle of treatment (taking capecitabine for at least four meals), 5 mL venous blood samples will be collected 2 hours after taking medicine in the morning, put into a tube with ethylene diamine tetraacetic acid (EDTA) anticoagulant, and sent to the pharmacy department of the hospital for testing immediately.
Adverse effects, including nausea, vomiting, diarrhea, leukopenia, anemia, thrombocytopenia, diarrhea, oral mucosal reaction, peripheral neuropathy, hand-foot syndrome, pigmentation, and abnormal liver function etc., will be assessed in each chemotherapy cycle using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 . All adverse effects will be recorded from grades I to IV according to severity.
In order to minimize the interference of personal diet on the gut microbiota and record changes in dietary habits in time, volunteers will be asked to complete a 24-hour food record each time a stool sample is collected. They need to describe all foods and beverages consumed in detail including ingredients, weight, and cooking style. Daily nutrient intakes will be calculated based on the China Food Composition 2009 .
QoL will be assessed in each chemotherapy cycle using Chinese version of QoL questionnaire-caner 30 (QLQ-C30) formulated by the European Organization for Research and Treatment of Cancer. The questionnaire has 30 items, including 5 functional scales, 3 symptom scales, 6 individual measures items, and a global health .
The prevalence of anxiety and depression in CRC patients is 13%-57%, and one of the important factors is the adverse effects of chemotherapy . Hospital Anxiety and Depression Scale (HADS), an effective questionnaire with 14 items, will be used to assess depression and anxiety in each chemotherapy cycle. Each item is on a 4-point scale and final scores are proportional to the degree of anxiety and depression.
Sample size calculation
Since there is no clinical trial assessing the effect of intestinal flora on the bioavailability of chemotherapy drugs and the intervention endpoints of intestinal flora are currently unable to determine, the sample size is estimated based on the data from a previous clinical trial  which assessed the effects of synbiotics intervention on chemotherapy-related adverse effects (including diarrhea, appetite loss, nausea, and vomiting) for CRC patients received post-operative chemotherapy, showing that 20 volunteers per group had 90% power at an alpha level of 0.05 to detect significant differences. Considering the long duration of the study (3 months) and the expected withdrawal of participants during the intervention, we plan to recruit n = 25 participants per group (n = 100 total).
The visiting time of the participants is in accordance with the treatment course, so as to facilitate the retention of the participants. The measured data for each visit will be collected on paper and then recorded electronically on a secure, locked office computer. The paper version of the data will be locked in a bookcase. Only the investigators running in the study will have access to the final study dataset.
The obtained data will be analyzed by SPSS software V26.0 (IBM, USA). Continuous data will be expressed as mean ± standard deviation (SD), and categorical data will be expressed as the number of cases (n) and percentage (%). Differences in parameter continuous variables and asymmetric variables between groups will be analyzed through independent samples t test and Mann-Whitney U test, respectively. Using a paired t test to assess the effect of the intervention in each group. The normality of data will be assessed using the Kolmogorov-Smirnov test.
Results will be regarded as statistically significant if p values< 0.05.