PREBICC - Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: protocol of a double-blind randomized controlled trial

PREBICC - Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: protocol of a double-blind randomized controlled trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Study protocol PREBICC - Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: protocol of a double-blind randomized controlled trial Lars E. Hillege, Milou A.M. Stevens, Judith de Vos-Geelen, Liselot Valkenburg-van Iersel, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8436668/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Standard systemic treatment for advanced colorectal cancer (CRC) often involves fluoropyrimidines, such as 5-fluorouracil (5-FU). Previous observational clinical studies, as well as pre-clinical research, demonstrated that chemotherapy affects the gut bacteria and its metabolites, potentially leading to microbial dysbiosis. Microbial dysbiosis might negatively influence anti-cancer efficacy and toxicity of the treatment. Therefore, targeted microbiota modulation using prebiotic fibers could potentially optimize 5-FU-based chemotherapy. This explorative intervention study aims to investigate the effects of daily administration of a prebiotic fiber mixture in a nutritionally complete oral nutritional supplement (ONS) on the intestinal microbiota composition in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy. Moreover, the effect of the prebiotic fiber ONS intervention on fecal, blood, clinical, and tolerance parameters will be evaluated. Methods 62 CRC patients scheduled for 5-FU-based therapy (FOLFOX, CAPOX, or capecitabine monotherapy) with or without bevacizumab, panitumumab or cetuximab will be prospectively enrolled in a double-blinded randomized controlled study. After informed consent, patients will be randomized to receive either the prebiotic fiber mixture ONS or control ONS, starting at least three days prior to the start of the 5-FU-based treatment. They will continue to take it twice daily for the duration of either four cycles of FOLFOX (in total 8 weeks) or three cycles of CAPOX or capecitabine monotherapy (in total 9 weeks). At multiple timepoints throughout the intervention, patients will collect fecal samples and complete questionnaires on food habits, patient characteristics, chemotherapy side effects, quality of life, current nutritional status, and physical performance. Microbiota composition, SCFA levels, and other parameters will be measured in the fecal samples. In addition, blood samples will be collected and analyzed for various parameters. Body composition and tumor response will be evaluated based on imaging. Discussion In conclusion, this randomized exploratory clinical intervention study outlines a novel approach to enhance the efficacy and reduce the toxicity of 5-FU-based chemotherapy in patients with advanced CRC through gut microbiota modulation using prebiotics. The knowledge obtained holds the potential to significantly impact clinical practices, improve patient outcomes, and advance microbiome-targeting interventions. Trial registration This study is registered in the Dutch OMON database under NL86537.068.24 Colorectal neoplasms Research design Prebiotics Microbiota Figures Figure 1 Figure 2 Administrative information Title PREBICC: Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: a randomized controlled clinical intervention study Trial registration Overview of medical research in the Netherlands (OMON) NL86537.068.24 Protocol version Version 1.4 Funding TKI Agri & Food (LWV20.345) Danone Global Research & Innovation Center Author details Lars E. Hillege 1,2 , Milou A.M. Stevens 1,2 , Judith de Vos-Geelen 1,3 , Liselot Valkenburg-van Iersel 1,3 , Sander M.J. van Kuijk 4 , Sander S. Rensen 2,5 , Guus Roeselers 6 , Ardy van Helvoort 5,6 , John Penders 5,7 , Marjolein L. Smidt 1,2 , Janine Ziemons 1,2 1 GROW - Research Institute for Oncology and Reproduction, Maastricht University, Maastricht, the Netherlands 2 Department of Surgery, Maastricht University Medical Center+, Maastricht, the Netherlands 3 Department of Internal Medicine, Division of Medical Oncology, Maastricht University Medical Center+, Maastricht, the Netherlands 4 Department of Clinical Epidemiology and Medical Technology Assessment, Maastricht University Medical Center+, Maastricht, the Netherlands. 5 NUTRIM - Institute of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands 6 Danone Research & Innovation, Utrecht, the Netherlands 7 Department of Medical Microbiology, Infectious Diseases and Infection Prevention, Maastricht University Medical Center+, Maastricht, the Netherlands Name and contact information for the trial sponsor Maastricht University GROW - Research Institute for Oncology and Reproduction, Maastricht University Role of sponsor and funder Role of sponsor: study design, data collection, lab analysis, data management, data analysis, and interpretation of data, writing of the scientific manuscript, and the decision to submit the manuscript for publication, authority over any of these activities. Role of funders: TKI Agri & Food (LWV20.345): Not applicable Danone Research & Innovation: study design, lab analysis, and providing feedback on the scientific manuscript. Roles and responsibilities of the research team Role and responsibilities p rincipal investigator: Design and conduct of the PREBICC study, preparation of protocol and revisions, completion of electronic CRF [case report forms], publication of study reports, and agreement of final protocol. Role and responsibilities c oordinating researcher: Design and conduct of the PREBICC study, preparation of protocol and revisions, preparation of investigators brochure (IB) and electronic CRFs [case report forms], publication of study reports, agreement of final protocol, recruitment of patients and liaising with principle investigator, reviewing progress of study and if necessary agreeing changes to the protocol and/or IB to facilitate the smooth running of the study, study planning, provide annual report to ethics committee, SAEs [Serious adverse events] reporting to ethics committee and in case of study product related SAEs Danone Research & Innovation, responsible for trial master file, budget administration, randomization, and organization of fecal and serum sample collection. Role and responsibilities s tudy monitor from Clinical Trial Center Maastricht (CTCM): Study monitoring according to the monitoring plan and data verification. Introduction Background and rationale Colorectal cancer (CRC) is the third most common cancer in the world (1). In case of (advanced) CRC, the standard systemic treatment is often based on fluoropyrimidines, such as 5-fluorouracil (5-FU). Despite the widespread use of 5-FU-based chemotherapy, there are two significant drawbacks. First, only a limited percentage of patients responds to the treatment. Previous research showed that approximately 35% of patients with advanced CRC responded to capecitabine (an oral prodrug of 5-FU) or 5-FU (2). Second, many patients experience toxicity during 5-FU-based treatment, which might require dose adjustments or even discontinuation of the therapy (3). For instance, a study of 181 patients receiving 5-FU-based treatment for colorectal cancer showed that during the first treatment cycle, 80% of the patients experienced toxicity, mainly mucositis, diarrhea, vomiting, or neutropenia (4). These disadvantages emphasize the relevance of new treatment strategies to optimize efficacy, reduce toxicity, and improve the quality of life (QoL) during 5-FU-based chemotherapy. The gut microbiota represents a promising target to achieve this, since it has been shown to interact with chemotherapeutic drugs and might affect both chemotherapy efficacy and toxicity (5-7). Previous research showed that 5-FU impacts the growth of specific gut bacteria (8-10), which could potentially contribute to a pro-inflammatory microbial milieu and the occurrence of increased (gastrointestinal) toxicity. In addition, we recently reported that capecitabine monotherapy reduces fecal levels of the bacteria-derived short-chain fatty acids (SCFA) valerate and caproate (11). Valerate has been shown to cause histone hyperacetylation and growth inhibition in human carcinoma cells and might thus play a role in anti-tumor efficacy (12). Furthermore, baseline fecal levels of the branched-chain fatty acid (BCFA) iso-butyrate were significantly lower in patients with partial response compared to patients with stable or progressive disease, indicating another potential link between the gut microbiota and response to 5-FU-based treatments (11). In line with this, recent in-vitro studies demonstrated that certain bacteria (e.g. E. coli ) are able to directly metabolize 5-FU, with negative consequences for its anti-tumor efficacy (13, 14). The studies of An et al. , and Yuan et al. also suggested that 5-FU efficacy is at least partly modulated by intestinal bacteria (15, 16). Recently, a pilot study investigating the effects of fecal microbiota transplantation (FMT) before chemotherapy found that FMT improved the disease control rate, as well as survival, in patients with advanced gastroesophageal cancer (17). In addition to treatment efficacy and toxicity, cancer cachexia is a factor that significantly influences the patient’s prognosis and quality of life. Different associations between the intestinal microbiota and cancer cachexia have been described (as reviewed in (18)). In a previous clinical study, we reported altered abundance of specific taxa and reduced levels of the SCFA acetate in cachectic cancer patients compared to non-cachectic cancer patients and healthy controls (19). Furthermore, cachexia-associated alterations in intestinal microbiota composition can be found, commonly characterized by an overgrowth of taxa belonging to the pro-inflammatory Enterobacteriaceae family (20-22). From the studies described above, it can be concluded that targeted modulation of the intestinal microbiota has the potential to optimize 5-FU-based chemotherapy by increasing therapy efficiency, decreasing toxicity, and reducing cancer cachexia. In this context, the principal aim would be to stimulate the growth of gut bacteria with beneficial properties, thereby counteracting the overgrowth of bacteria with pro-inflammatory properties and/or an unwanted role in 5-FU metabolism. To achieve this, prebiotics are a promising strategy.Prebiotics are substrates that are selectively fermented by microorganisms and confer a health benefit to the host, by stimulating the growth and/or activity of beneficial bacteria (23). In addition, prebiotic fermentation leads to the formation of SCFA, which not only modulate the growth of cancer cells (as described above), but also have potent anti-inflammatory as well as beneficial metabolic effects (24-26). Supplementation with the prebiotic fructo-oligosaccharide (FOS) in a mouse model of 5-FU-induced mucositis improved gut integrity by enhancing SCFA production, maintaining tight junctions, and reducing inflammation (27). In previous in-vitro experiments, we found that prebiotic fiber mixtures also exert these positive effects in the presence of 5-FU (28). As a next step, the PREBICC study aims to investigate whether these beneficial effects can also be achieved in CRC patients during 5-FU-based chemotherapy and whether these microbiota alterations improve 5-FU toxicity and/or efficacy as well as cachexia. Objectives This study aims to investigate the effects of daily administration of a prebiotic fiber mixture in a nutritionally complete Oral Nutritional Supplement (ONS) on the abundance of microbial taxa with beneficial properties (e.g. Bifidobacterium ), as well as on intestinal microbiota composition and diversity, compared to a control ONS, in patients with advanced CRC treated with 5-FU-based chemotherapy. As exploratory objectives, this study will assess (1) thefeasibility, tolerance, and compliance of a nutritional intervention with a prebiotic ONS in patients with advanced CRC, (2) the effects of the prebiotic ONS on fecal and blood parameters, tumor response, chemotherapy toxicity as well as the metabolic phenotype (e.g. body weight, body composition, inflammatory profile), and (3) the relationship with the patient-reported QoL and physical functioning. Trial design This intervention study is a double-blind randomized controlled trial in patients with advanced CRC receiving 5-FU-based chemotherapy and will be performed in the Maastricht University Medical Center (MUMC+), the Netherlands, with the potential for future expansion to a multicenter setting. Patients will be stratified based on their assigned chemotherapy regimen (FOLFOX, CAPOX, or capecitabine monotherapy) before 1:1 randomization. The block size will be either 2 or 4. Patients will be randomized to either the test group (ONS enriched with prebiotic fibers) or the control group (isocaloric, isonitrogenous ONS without prebiotic fibers). Patients will start with the prebiotic or control ONS at least three days prior to the start of the 5-FU-based treatment. They will continue to take it twice daily for the duration of either four cycles of FOLFOX (in total 8 weeks) or three cycles of CAPOX or capecitabine monotherapy (in total 9 weeks). Fecal samples will be collected at multiple time points throughout the study period: before the start of the intervention (T0) and in the last two days of chemotherapy cycles 1 (T1), 2 (T2), 3 (T3), and 4 (T4, for FOLFOX only). Simultaneously, patients will fill in questionnaires concerning amongst others body weight and length, experienced side effects of chemotherapy (according to the Common Terminology Criteria for Adverse Events (CTCAE)), gastrointestinal tolerance and stool consistency (Bristol stool scale), QoL (EORTC QLQ-C30 (29) and EQ-5D-5L (30)), current nutritional status (Patient-Generated Subjective Global Assessment (PG-SGA) score), and physical performance (Karnofsky Performance Score (KPS)). A food habits questionnaire (FQ18) will be completed at the start of the study period (T0) (31). In addition, repeated blood samples will be collected to analyze amino acids, C-reactive protein (CRP), and SCFA concentrations. Changes in dietary habits, compliance, and changes in patients’ body weight will be recorded throughout the study period. Routine CT/MRI scans before and during the third or fourth chemotherapy cycle will be used to evaluate body composition (CT scans only), as well as tumor response (based on the ‘Response Evaluation Criteria in Solid Tumors’ (RECIST) (32)) (Figure 1). Methods: Participants, interventions, and outcomes Eligibility criteria The study population includes patients diagnosed with advanced CRC who are scheduled for treatment with 5-FU-based therapy (FOLFOX, CAPOX, or capecitabine monotherapy). Simultaneous treatment with bevacizumab, panitumumab, or cetuximab is permitted, if no systemic antibiotics are used (topical antibiotics are allowed). A full list of all the inclusion and exclusion criteria is provided in Table 1. Table 1: Inclusion and exclusion criteria Inclusion criteria Patients diagnosed with advanced CRC who will be treated with 5-FU-based therapy (FOLFOX, CAPOX, capecitabine monotherapy) Simultaneous treatment with bevacizumab, panitumumab, or cetuximab is allowed, provided that no systemic antibiotics are used (topical antibiotics are allowed) Proficient use of the Dutch language The patient is older than 18 years of age Performance (ECOG/WHO) score 0-2 (33) Written informed consent Exclusion criteria Microsatellite instability (MSI) or deficient mismatch repair (MMR) proteins Known abnormal DPYD variants (single nucleotide polymorphisms) and/or reduced dihydropyrimidine dehydrogenase (DPD) enzyme function Presence of ileostomy Pregnant or nursing Previous systemic therapy for advanced CRC. If the patient received prior (neo)adjuvant systemic therapy, it must have been completed at least 6 months before the diagnosis of the advanced disease Therapeutic systemic antibiotics used less than four weeks before the start of 5-FU-based therapy (topical antibiotics are allowed) Abdominal radiotherapy less than two weeks before the start of the 5-FU-based therapy Use of pro- and/or prebiotics during the study period Inflammatory bowel disease (Crohn’s disease or ulcerative colitis) Simultaneous treatment with irinotecan chemotherapy Known allergy to any ingredients present in the test or control ONS, requiring a fiber-free diet, or suffering galactosemia or lactose intolerance Simultaneous participation in another medical-scientific intervention study Physically or mentally incapable or incompetent All conditions that, in the opinion of the physician, are not suitable for participation in this study (e.g. severe renal failure). Intervention Description The intervention for this study involves twice daily consumption of an ONS with or without the addition of prebiotic fibers for eight or nine weeks depending on the chemotherapy regimen. Patients will start the ONS intake at least three days prior to the start of 5-FU-based chemotherapy. The test and control ONS are nutritionally complete, high-energy (300 kcal), high-protein (12g), ready-to-drink supplements, intended for medical use (Nutricia, Zoetermeer, The Netherlands). The test ONS additionally contains 4.5g of fibers, a mixture of galacto-oligosaccharides (GOS), FOS, and pectin per bottle. Both the test and control ONS are supplied in blinded 125 mL bottles and will be available in two flavors: vanilla and strawberry (Appendix 1, supplementary table 1). Modifications Different modifications to the study procedure are possible. First of all, patients are free to withdraw from the study at any time and without any consequences. Secondly, the intervention period might be prolonged if the medical oncologist adapts the 5-FU treatment schedule of a patient. If the aggregated total treatment delay throughout the study period exceeds four weeks, study participation of this patient will be discontinued after the collection of a final fecal sample. Thirdly, if a patient requires additional calories to maintain weight during chemotherapy, the dosage of the test/control ONS will be increased to meet the energy requirements. Furthermore, in case of non-compliance, the researcher can discontinue participation of a patient in the trial. If a patient experiences study product-related side effects that cause significant harm or make continued participation impractical, the researcher may choose to withdraw the patient from the study, especially in the case of a serious adverse event (SAE). Adherence Patients are regularly interviewed concerning their consuming experiences and should return any unconsumed drinks at the visits with the researcher at the start of each cycle during the study period. This information will be used to monitor compliance. To increase adherence, patients can choose one or a combination of the flavors: vanilla or strawberry. Outcomes Main outcomes The main outcomes include the differences in shifts (∆baseline vs end of study) of the relative abundance of specific microbial taxa (e.g. Bifidobacterium ), as well as of overall intestinal microbiota composition and diversity between test and control group. Exploratory outcomes Longitudinal changes in: Parameters assessed in feces Intestinal microbiome composition parameters: e.g., alpha & beta diversity and taxonomic and functional composition during all timepoints Functional capacity of the intestinal microbiota during all timepoints Levels of SCFA and BCFA pH and dry weight Concentrations of measures of gut inflammation (calprotectin), immune status and gut integrity (secretory immunoglobulin A (sIgA)) and microbial metabolism (ammonia) (34-36) Parameters assessed in blood Amino acids concentrations C-reactive protein (CRP) concentrations Levels of SCFA Clinical parameters Nutritional parameters: body weight, BMI, and PG-SGA score (37) Dietary habits: FQ18 questionnaire and follow-up questionnaires during the study (31) Experienced toxicity related to chemotherapy: CTCAE questionnaire (38) Physical performance: Karnofsky performance score (39) Quality of life: EORTC QLQ-C30 and EQ-5D-5L (29, 30) Body composition: measured using routine CT scan at L3 level Tumor response: RECIST criteria (32) Tolerance parameters Prebiotic and control ONS compliance Gastrointestinal tolerance: Bristol stool scale and diarrhea prevalence (40) Other outcomes In addition to the main and secondary outcomes, the following patient characteristics will be recorded: age, sex, pre-study weight loss in the last six months, and medication and supplement usage before and during the study. In addition, information concerning medical history and hematological parameters, i.e. leukocytes, neutrophils, and thrombocytes, will be retrieved from medical records. Additional fecal and blood samples will be stored to enable potential future analysis of other exploratory outcomes, for instance fecal levels of tryptophan (metabolites) and D-Amino acids in feces or blood cytokine levels. Participant timeline An overview of the study timeline is depicted in Figure 2. Sample size The main outcome of this study is the relative abundance of members of the genus Bifidobacterium (log10 copies/gram wet weight feces) in fecal samples, measured at baseline (pre-5-FU-based chemotherapy and pre-ONS-intervention) and post-intervention between the test and control group. This primary endpoint involves comparing the mean change from baseline (post-baseline minus baseline) between test and control group. The difference between groups will be evaluated using the independent-samples t-test or linear regression models. The sample size calculation is based on estimates from a prior study using a similar prebiotic ONS (OMON registration number NL1693). Based on this study, a standard deviation of 0.35 is assumed. The smallest detectable difference with sufficient power is set to 0.34. Based on the provided estimates and parameters, a sample size of 46 patients, with 23 in each group (test and control), is considered sufficient to detect a statistically significant difference in Bifidobacteria changes between the test and control groups using a two-sided independent-samples t-test, when testing with a significance level (α) of 0.05, and a power of 90%. Considering an anticipated dropout rate of 25% for the intervention period, the total number of subjects to be enrolled is adjusted to 62, with 31 patients in each of the two groups (intervention and control). This adjustment ensures that even with the expected dropouts, there will be a sufficient number of patients to maintain the statistical power of the study as planned. The sample size calculation was performed using the formula from Chow et al . with R software for statistical computing (41). Recruitment Medical oncologists (in training) at the MUMC+ will ask eligible patients for their consent to be contacted by the researcher and/or will briefly inform the patient about the study. Subsequently, the researcher will further inform the patient about the aims of the study, explain the sampling procedure, and expectations throughout the intervention. A patient information letter, a flyer containing all essential information about the study, and informed consent form will be provided. After a reflection time of at least 24 hours up until 7 days when treatment schedule and anticipated start of treatment allows, patients are asked if they would like to participate. If the patient agrees to proceed, an appointment with the researcher is scheduled and informed consent (Appendix 2) will be signed together. Methods: Assignment of interventions Allocation Eligible patients will be allocated to one of two groups: test or control group. To ensure balance across these two groups regarding key prognostic variables, patients will be stratified based on their assigned chemotherapy regimen prior to randomization (FOLFOX, CAPOX, capecitabine monotherapy). Following stratification, patients will undergo a 1:1 randomization with block sizes of either 2 or 4 to ensure an equal number of patients in each group. The randomization process will be facilitated using Castor and ALEA systems. ALEA will automatically assign each patient to either the test or control group, adhering to a predefined block randomization scheme. Following the assignment, ALEA selects a product number from a predetermined list, which is managed by an unblinded researcher in collaboration with the supplies manager. This list comprises product numbers uniquely associated with either the prebiotic or control ONS, ensuring that patients receive the correct ONS according to their group assignment. This ensures that blinded researchers, even after an unblinding event, will not be able to ascertain the group assignments of other patients within the study. The study coordinator will perform the generation of the allocation sequence, the enrollment of patients, and the assignment of patients to interventions. Blinding Neither the patients nor the researchers know which ONS is administered until the clinical trial concludes. Only one independent researcher and the supplies manager, who are not involved in patient contact, sample processing, or data analysis, will be unblinded. Emergency unblinding of the allocation will occur if patients experience SAEs which are likely to be related to the study product. Methods: Data collection, management, and analysis Data collection plan Fecal samples Fecal samples will be collected at several time points throughout the study period. Patients are asked to collect the fecal samples at home in preservation-free tubes (Sarstedt) and to immediately store them in the freezer. The samples will be transported to the hospital in a cooled container (Sarstedt) to prevent thawing and will be stored at -80 °C. Metagenomic sequencing will be performed to quantify fecal microbiota composition, diversity, functional capacity, and taxonomic abundances. To this end, total DNA will be extracted from fecal samples using the QIAmp DNA Stool Mini Kit (Qiagen) where the manufacturer’s protocol will be followed but with an addition of two bead-beating steps. The Illumina Nextera XT DNA Library Prep kit (Illumina, San Diego, USA) will be used as per the manufacturer’s instructions. Library quality control will be checked, and the samples will be loaded onto the NovaSeq 6000 platform. Subsequently, metabolic activity of the gut microbiota will be assessed. The pH, SCFA levels (i.e. acetate, propionate, butyrate, valerate), BCFA levels (iso-butyrate, iso-valerate), ammonia, and calprotectin concentrations will be measured. Details on the methods that will be used to measure these physiological parameters have been described previously (42, 43). For the assessment of sample dry weight, 500mg of frozen feces will be dried in a vacuum dryer (Eppendorf) for five hours and will be weighted afterwards. Blood samples Blood samples will be collected, if possible together with routine blood samples, before every cycle during the intervention period. The concentrations of amino acids, CRP, and levels of SCFA will be measured. Questionnaires During the intervention period, patients will complete the following questionnaires. Patient-Generated Subjective Global Assessment (PG-SGA) is a patient-reported questionnaire for assessment of the nutritional status in cancer patients (37). In the current study, the Short Form version will be used to assess the current nutritional status at the same time as fecal sample collection. FQ18 is a Dutch web-based food habits questionnaire developed by Wageningen University & Research. It assesses intake of energy, macronutrients, type of fatty acids and dietary fiber (31). The FQ18 will be completed only once at the beginning of the study period. Common Terminology Criteria for Adverse Events (CTCAE) will be used to assess chemotherapy side effects during each cycle in the study period, e.g. nausea and diarrhea (38). Karnofsky Performance Score (KPS) will be used to assessphysical performance as this validated questionnaire assesses physical and psychosocial difficulties (39). EORTC QLQ-C30 is widely used to assess the QoL in cancer patients, including those with colorectal cancer. Developed by the European Organisation for Research and Treatment of Cancer (EORTC), it measures various aspects of QoL, such as physical, emotional, cognitive, and social functioning, as well as symptom burden. For colorectal cancer patients, relevant items may include issues related to eating, fatigue, pain, and gastrointestinal symptoms (29). EQ-5D-5L is a standardized measure of health-related QoL, assessing five dimensions of health with five severity levels each (30). Bristol Stool Scale was developed in 1997 for the purpose of evaluating intestinal transit time.Utilization of this scale enables the assessment of gastrointestinal tolerance, as alterations in bowel habits resultant from the intervention can be monitored (40). Body composition and tumor response CT or MRI scans will be made for routine care before the intervention and during the third or fourth treatment cycle. These CT scans will be used to evaluate body composition (based on the image at the third lumbar vertebra (L3) level) and CT or MRI scans will be used to evaluate tumor response (based on the RECIST criteria (32)). Additional clinical information Clinical information from the questionnaires will be completed with information derived from the patient’s medical record, for instance concerning toxicity, dose adjustments, medical history, and co-medication. In addition, the blood parameters, as well as parameters for liver and kidney function will be assessed in the context of standard care and retrieved from the patient’s medical record. Follow up after participation withdraw When a patient decides to withdraw from the study, any unused study products and other study materials will be returned in consultation with the researcher. Approximately two weeks after taking the last study product, patients will be contacted to discuss any delayed side effects and to evaluate their participation in the intervention study. Data management Personal data will be handled with strict care securing the patients' privacy according to the EU General Data Protection Regulation. All patients and their samples receive a pseudonymized study code. The key linking study code to patient data (hospital ID and name) will only be accessible to the primary researcher as well as the (coordinating) researcher(s). Electronic data will be safely stored and backed up in an electronic data capture system. The research process will be documented. Data validation messages are implemented to provide warnings for potential errors in the data input. Additional quality checks will be performed before, during and after the data collection. All study samples will be centrally stored at the Biobank of the MUMC+ until used for analysis. Statistics Statistical analyses will be performed with R and R Studio. All analyses will be performed according to the intention to treat principle. In case of missing data in over 5% of patient records, an imputation method will be chosen depending on the extent of missing data and the likely missing data mechanism(s). Main outcomes Raw data from metagenomic sequencing will be processed further and analyzed using state-of-the-art in-house data analysis pipelines. The main study parameter is focused on exploring differences in the relative abundance of specific bacterial taxa (∆baseline vs end of study) between the test group and control group. For this purpose, differential abundance analyses, which were specifically developed for compositional data (CoDA) will be used. Furthermore, generalized linear mixed-effects models on centered log ratio (CLR) transformed microbial counts will be used for repeated measurements. Overall intestinal microbiota composition (β-diversity) and clustering of samples will be visualized by means of Principal Component Analysis (PCA). The Shannon index and observed species richness will be calculated as measures of microbial diversity (α-diversity). Intention-to-treat analyses will be performed. Secondary outcomes To determine whether parametric or nonparametric tests should be performed, normality will be assessed for all variables based on histograms, Q-Q-Plots, and the Shapiro-Wilk test. Longitudinal analysis (changes during the study period) will be conducted using Friedman’s ANOVA or linear mixed-effects models. For cross-sectional analyses comparing the intervention and control group at specific time points, the Kruskal–Wallis or Mann–Whitney U test will be used for non-normally distributed data, and the t-test or ANOVA if the assumption of normality is met. To analyse associations between different clinical variables of interest, Spearman correlation will be computed, and p-values will be adjusted for multiple testing by means of False Discovery Rate (FDR) adjustments according to the Benjamini and Hochberg procedure (44). Permutational multivariate analysis of variance (PERMANOVA) will be performed to assess the correlation between microbial profiles and (clinical) determinants as it is a non-parametric method and it accommodates the complex nature of microbial community data. Methods: Monitoring Data monitoring The study will be monitored by the Clinical Trial Center Maastricht (CTCM), according to a predefined monitoring plan. The monitor will be independent from the sponsor of this study. There will be no Data and Safety Monitoring Board (DSMB) installed, since the product under investigation has a history of safe use and is not expected to cause severe undesired side effects. Harms All adverse events (AE) that occur during the study period will be recorded, whether or not considered related to the intervention. Furthermore, the researcher will report SAEs to the sponsor, directly after obtaining knowledge of the events, except for the following SAEs that occur commonly in the patient population under investigation: Elective hospitalization or hospitalization related to technical, practical, or social reasons (i.e. prolonging hospital stay to arrange aftercare) Hospitalization due to common side effects of the chemotherapeutic regimen assessed by the treating physician Events as a result of the progression of the disease under study. SAEs will be reported through the web portal ToetsingOnline to the METC azM/UM. In case of SAEs considered to be related to the use of the study product, the supplier Danone Research & Innovation will be informed. Discussion Fluoropyrimidine-based treatments, such as 5-FU and capecitabine, either in combination with oxaliplatin or irinotecan, form the backbone of advanced CRC therapy (45), although only part of the patients respond well (2). Additionally, many patients experience significant toxicity, necessitating dose adjustments or discontinuation, and further decreasing 5-FU efficacy (3). These limitations highlight the need for new strategies to optimize efficacy, reduce toxicity, and improve QoL during 5-FU-based chemotherapy. Since the gut microbiota has been shown to interact with 5-FU and might impact its efficacy and toxicity, the PREBICC study aims to investigate whether an ONS enriched with prebiotic fibers could stimulate abundance of bacteria with beneficial properties and modulate gut microbiota composition and diversity in patients with advanced CRC treated with 5-FU-based chemotherapy. Furthermore, the study aims to explore whether microbiota alterations correlate with improvements in 5-FU toxicity, tumor response, physical functioning, metabolic phenotype, and QoL, as well as evaluates the feasibility of such an intervention. While there are also other on-going studies using prebiotics in cancer patients (46, 47), most trials currently focus on probiotics or FMT (48). We decided to test prebiotics, because they do not contain living bacteria, are considered to induce more holistic microbiota changes compared to probiotics, and are less invasive compared to FMT. Since different prebiotic fibers might exert distinct effects depending on their physicochemical properties (49, 50), we chose the prebiotic ONS based on our previous in-vitro experiments (28). Furthermore, an additional strength of this clinical trial is its randomized double-blinded design. The potential implications of this study are substantial. If daily administration of a prebiotic ONS proves effective in modulating the gut microbiota, enhancing chemotherapy efficacy, and reducing toxicity, it could lead to significant improvements in the standard treatment protocols for advanced CRC. By optimizing gut microbiome composition, patients may experience fewer side effects, allowing for more consistent dosing and potentially better overall outcomes. Furthermore, a reduction in cancer cachexia could greatly improve patients' QoL and prognosis. Additionally, PREBICC will provide important insights into the feasibility of gut microbiota modulation during chemotherapy and the specific needs of patients with advanced cancer. Altogether, these results will greatly contribute to the tailor-made and personalized design of prebiotic interventions and implementation into clinical practice. There are several limitations to the current study. First, we previously reported considerable inter-individual variability in gut microbiota composition among CRC patients treated with capecitabine, most likely due to diverse medical histories and the administration of antibiotics and other co-medication (51). We are unable to exclude that this will also occur in the PREBICC cohort, which could complicate the detection of 5-FU or prebiotic-induced effects. Secondly, the efficacy of the intervention mainly depends on compliance of the patients (52). Reliable assessment of patient compliance is challenging, and the methods used in this study can only estimate compliance. In conclusion, the PREBICC study protocol outlines a novel approach to enhance the efficacy and reduce the toxicity of 5-FU-based chemotherapy in patients with advanced CRC through the modulation of the gut microbiota using prebiotics. This exploratory research holds the potential to significantly impact clinical practices and improve patient outcomes, paving the way for future studies and advancements in microbiome-based therapies. Abbreviations Abbreviation Definition 5-FU 5-fluorouracil AE Adverse events BCFA Branched-chain fatty acids CCMO Centrale Commissie Mensgebonden Onderzoek CLR Centered log ratio CoDA Compositional data CRC Colorectal cancer CRFs Case report forms CRP C-reactive protein CTCAE Common Terminology Criteria for Adverse Events CTCM Clinical Trial Center Maastricht DPDD Dihydropyrimidine dehydrogenase DSMB Data and Safety Monitoring Board FDR False Discovery Rate FMT Fecal microbiota transplantation FOS Fructo-oligosaccharide GOS Galacto-oligosaccharides IB Investigators brochure ICH-GCP International Conference on Harmonization Good Clinical Practice ICMJE International Committee of Medical Journal Editors KPS Karnofsky Performance Score MMR Mismatch repair MSI Microsatellite instabillity MUMC+ Maastricht University Medical Center+ OMON Overview of medical research in the Netherlands ONS Oral nutritional supplement PCA Principal Component Analysis PERMANOVA Permutational multivariate analysis of variance PG-SGA Patient-Generated Subjective Global Assessment QoL Quality of life RECIST Response Evaluation Criteria in Solid Tumors SAE Serious adverse event SCFA Short-chain fatty acids sIgA Secretory immunoglobulin A Declarations Ethics and dissemination Research ethics approval This study was approved by the Medical Ethical Committee azM/UM (NL86537.068.24 / METC24-011) and Institutional Review Board of Maastricht UMC+. The study will be conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki and consistent with the International Conference on Harmonization Good Clinical Practice (ICH-GCP). Protocol amendments Any essential changes in the study protocol and associated documents will be submitted to the medical ethical committee for assessment. If changes need to be implemented, this will be communicated to the parties involved. Consent Patients will be informed in detail about the study aims and procedures. After a reflection time, patients are asked whether they would like to participate or not and informed consent will be signed in the presence of the researcher. Declaration of interests LEH, MAMS, JZ and MLS have received research funding from Danone Research & Innovation. AvH is employed by Danone Research & Innovation, GR was employed by Danone Research & Innovation during the development of the study protocol. MLS has also received funding from Servier and Illumina, all outside the submitted work. JVG has served as a consultant for Amgen, AstraZeneca, MSD, Pierre Fabre, and Servier, and has received institutional research funding from Servier, all outside the submitted work. LVI has served as a consultant for Amgen, MSD, Pierre Fabre, and Servier. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data access All data handling procedures will adhere to the regulations outlined in the EU General Data Protection Regulation and the Dutch Act on Implementation of the General Data Protection Regulation. F unding This research was funded by TKI Agri & Food (LWV20.345) and Danone Global Research & Innovation Center. The funding bodies had no role in the study design, data collection, analysis, or interpretation of the data, nor in the writing of the manuscript. Dissemination policy The principal investigator will be responsible for the public disclosure/publication of the study outcome. Scientific findings will be published in open-access, peer-reviewed journals, adhering to the “Centrale Commissie Mensgebonden Onderzoek” (CCMO) guideline and the International Committee of Medical Journal Editors (ICMJE) criteria for co-authorship. Additionally, results will be disseminated at various forums, including stakeholder-specific conferences, symposia, workshops, and training events. Other platforms for sharing information will include social media, press releases, project websites, newsletters, and science weeks. Data used and/or analyzed will be available after completion of the study from the corresponding author on reasonable request. Raw sequencing data will be submitted to an online repository. Furthermore, the trial is registered in a public trial registry (OMON) and sequencing data obtained in the context of this study will be deposited in a public repository after the completion of the analyses. Au thor contributions LEH: Conceptualization, Methodology, Funding Acquisition, Project Administration, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing, Ethics Approval, Data Curation, Validation, Visualization. MAMS: Conceptualization, Methodology, Project Administration, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing, Ethics Approval, Data Curation, Validation, Visualization. JVG: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review & Editing, Data Curation, Validation. LVI: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review & Editing, Data Curation, Validation. SMJK: Conceptualization, Methodology, Writing - Review & Editing, Validation. SSR: Conceptualization, Funding Acquisition, Writing - Review & Editing. GR: Conceptualization, Funding Acquisition, Writing - Review & Editing. AH: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review & Editing, Validation. JP: Conceptualization, Methodology, Funding Acquisition, Resources, Supervision, Writing - Review & Editing, Validation. MLS: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review & Editing, Data Curation, Validation. JZ: Conceptualization, Methodology, Funding Acquisition, Project Administration, Investigation, Resources, Supervision, Writing - Original Draft, Writing - Review & Editing, Ethics Approval, Data Curation, Validation, Visualization. References Rawla P, Sunkara T, Barsouk A. Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors. Prz Gastroenterol. 2019;14(2):89–103. Petrelli F, Cabiddu M, Barni S. 5-Fluorouracil or capecitabine in the treatment of advanced colorectal cancer: a pooled-analysis of randomized trials. Med Oncol. 2012;29(2):1020–9. Timmers L, Boons CC, Mangnus D, Van de Ven PM, Van den Berg PH, Beeker A, et al. Adherence and Patients' Experiences with the Use of Capecitabine in Daily Practice. Front Pharmacol. 2016;7:310. Gusella M, Crepaldi G, Barile C, Bononi A, Menon D, Toso S, et al. Pharmacokinetic and demographic markers of 5-fluorouracil toxicity in 181 patients on adjuvant therapy for colorectal cancer. Ann Oncol. 2006;17(11):1656–60. Alexander JL, Wilson ID, Teare J, Marchesi JR, Nicholson JK, Kinross JM. Gut microbiota modulation of chemotherapy efficacy and toxicity. Nat Rev Gastroenterol Hepatol. 2017;14(6):356–65. Aarnoutse R, Ziemons J, Penders J, Rensen SS, de Vos-Geelen J, Smidt ML. The Clinical Link between Human Intestinal Microbiota and Systemic Cancer Therapy. Int J Mol Sci. 2019;20(17). Wong SH, Yu J. Gut microbiota in colorectal cancer: mechanisms of action and clinical applications. Nat Rev Gastroenterol Hepatol. 2019;16(11):690–704. Maier L, Pruteanu M, Kuhn M, Zeller G, Telzerow A, Anderson EE, et al. Extensive impact of non-antibiotic drugs on human gut bacteria. Nature. 2018;555(7698):623–8. Sougiannis AT, VanderVeen BN, Enos RT, Velazquez KT, Bader JE, Carson M, et al. Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota. Brain Behav Immun. 2019;80:44–55. Carvalho R, Vaz A, Pereira FL, Dorella F, Aguiar E, Chatel JM, et al. Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP. Sci Rep. 2018;8(1):15072. Ziemons J, Aarnoutse R, Heuft A, Hillege L, Waelen J, de Vos-Geelen J et al. Fecal levels of SCFA and BCFA during capecitabine in patients with metastatic or unresectable colorectal cancer. Clin Exp Med. 2023. Hinnebusch BF, Meng S, Wu JT, Archer SY, Hodin RA. The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. J Nutr. 2002;132(5):1012–7. LaCourse KD, Zepeda-Rivera M, Kempchinsky AG, Baryiames A, Minot SS, Johnston CD, et al. The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota. Cell Rep. 2022;41(7):111625. Spanogiannopoulos P, Kyaw TS, Guthrie BGH, Bradley PH, Lee JV, Melamed J, et al. Host and gut bacteria share metabolic pathways for anti-cancer drug metabolism. Nat Microbiol. 2022;7(10):1605–20. An J, Ha EM. Combination Therapy of Lactobacillus plantarum Supernatant and 5-Fluouracil Increases Chemosensitivity in Colorectal Cancer Cells. J Microbiol Biotechnol. 2016;26(8):1490–503. Yuan L, Zhang S, Li H, Yang F, Mushtaq N, Ullah S, et al. The influence of gut microbiota dysbiosis to the efficacy of 5-Fluorouracil treatment on colorectal cancer. Biomed Pharmacother. 2018;108:184–93. de Clercq NC, van den Ende T, Prodan A, Hemke R, Davids M, Pedersen HK, et al. Fecal Microbiota Transplantation from Overweight or Obese Donors in Cachectic Patients with Advanced Gastroesophageal Cancer: A Randomized, Double-blind, Placebo-Controlled, Phase II Study. Clin Cancer Res. 2021;27(13):3784–92. Ziemons J, Smidt ML, Damink SO, Rensen SS. Gut microbiota and metabolic aspects of cancer cachexia. Best Pract Res Clin Endocrinol Metab. 2021:101508. Ubachs J, Ziemons J, Soons Z, Aarnoutse R, van Dijk DPJ, Penders J, et al. Gut microbiota and short-chain fatty acid alterations in cachectic cancer patients. J Cachexia Sarcopenia Muscle. 2021;12(6):2007–21. Bindels LB, Beck R, Schakman O, Martin JC, De Backer F, Sohet FM, et al. Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model. PLoS ONE. 2012;7(6):e37971. Bindels LB, Neyrinck AM, Claus SP, Le Roy CI, Grangette C, Pot B, et al. Synbiotic approach restores intestinal homeostasis and prolongs survival in leukaemic mice with cachexia. Isme j. 2016;10(6):1456–70. Pötgens SA, Brossel H, Sboarina M, Catry E, Cani PD, Neyrinck AM, et al. Klebsiella oxytoca expands in cancer cachexia and acts as a gut pathobiont contributing to intestinal dysfunction. Sci Rep. 2018;8(1):12321. Gibson GR, Hutkins R, Sanders ME, Prescott SL, Reimer RA, Salminen SJ, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol. 2017;14(8):491–502. van der Beek CM, Dejong CHC, Troost FJ, Masclee AAM, Lenaerts K. Role of short-chain fatty acids in colonic inflammation, carcinogenesis, and mucosal protection and healing. Nutr Rev. 2017;75(4):286–305. Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity. 2014;40(1):128–39. Morrison DJ, Preston T. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes. 2016;7(3):189–200. Carvalho PLA, Andrade MER, Trindade LM, Leocádio PCL, Alvarez-Leite JI, Dos Reis DC, et al. Prophylactic and therapeutic supplementation using fructo-oligosaccharide improves the intestinal homeostasis after mucositis induced by 5- fluorouracil. Biomed Pharmacother. 2021;133:111012. Ziemons J, Hillege LE, Aarnoutse R, de Vos-Geelen J, Valkenburg-van Iersel L, Mastenbroek J, et al. Prebiotic fibre mixtures counteract the manifestation of gut microbial dysbiosis induced by the chemotherapeutic 5-Fluorouracil (5-FU) in a validated in vitro model of the colon. BMC Microbiol. 2024;24(1):222. Aaronson NK, Ahmedzai S, Bergman B, Bullinger M, Cull A, Duez NJ, et al. The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Cancer Inst. 1993;85(5):365–76. Janssen MF, Bonsel GJ, Luo N, Is. EQ-5D-5L Better Than EQ-5D-3L? A Head-to-Head Comparison of Descriptive Systems and Value Sets from Seven Countries. PharmacoEconomics. 2018;36(6):675–97. Trijsburg L, de Vries JH, Boshuizen HC, Hulshof PJ, Hollman PC, van 't Veer P, et al. Comparison of duplicate portion and 24 h recall as reference methods for validating a FFQ using urinary markers as the estimate of true intake. Br J Nutr. 2015;114(8):1304–12. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228–47. Blagden SP, Charman SC, Sharples LD, Magee LR, Gilligan D. Performance status score: do patients and their oncologists agree? Br J Cancer. 2003;89(6):1022–7. Jukic A, Bakiri L, Wagner EF, Tilg H, Adolph TE. Calprotectin: from biomarker to biological function. Gut. 2021;70(10):1978–88. Rollenske T, Burkhalter S, Muerner L, von Gunten S, Lukasiewicz J, Wardemann H, et al. Parallelism of intestinal secretory IgA shapes functional microbial fitness. Nature. 2021;598(7882):657–61. Oliphant K, Allen-Vercoe E. Macronutrient metabolism by the human gut microbiome: major fermentation by-products and their impact on host health. Microbiome. 2019;7(1):91. Balstad TR, Bye A, Jenssen CR, Solheim TS, Thoresen L, Sand K. Patient interpretation of the Patient-Generated Subjective Global Assessment (PG-SGA) Short Form. Patient Prefer Adherence. 2019;13:1391–400. Common Terminology Criteria for Adverse Events (CTCAE) U.S.: National Institutes of Health _ National Cancer Institute. 2017 [Version v5.0:[Available from: https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm Pallialine. Karnosfky Performance Score 2016 [Available from: http://www.pallialine.nl/uploaded/docs/Depressie/Bijlage%20Karnofsky.pdf Lewis SJ, Heaton KW. Stool form scale as a useful guide to intestinal transit time. Scand J Gastroenterol. 1997;32(9):920–4. Chow S-C, Wang H, Shao J. Sample Size Calculations in Clinical Research. 2nd ed2007. Phavichitr N, Wang S, Chomto S, Tantibhaedhyangkul R, Kakourou A, Intarakhao S, et al. Impact of synbiotics on gut microbiota during early life: a randomized, double-blind study. Sci Rep. 2021;11(1):3534. Huet F, Abrahamse-Berkeveld M, Tims S, Simeoni U, Beley G, Savagner C, et al. Partly Fermented Infant Formulae With Specific Oligosaccharides Support Adequate Infant Growth and Are Well-Tolerated. J Pediatr Gastroenterol Nutr. 2016;63(4):e43–53. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc: Ser B (Methodol). 1995;57(1):289–300. Taşçı E, Oyan B, Sönmez Ö, Mutlu AU, Atcı MM, Sakin A, et al. Comparing the efficacy of regorafenib and 5-fluorouracil-based rechallenge chemotherapy in the third-line treatment of metastatic colorectal cancer. BMC Cancer. 2024;24(1):16. Chen Y, Liao X, Li Y, Cao H, Zhang F, Fei B, et al. Effects of prebiotic supplement on gut microbiota, drug bioavailability, and adverse effects in patients with colorectal cancer at different primary tumor locations receiving chemotherapy: study protocol for a randomized clinical trial. Trials. 2023;24(1):268. US National Library of Medicine ClinicalTrials. gov 2023 [Available from: https://clinicaltrials.gov/study/NCT05516641 Ciernikova S, Sevcikova A, Drgona L, Mego M. Modulating the gut microbiota by probiotics, prebiotics, postbiotics, and fecal microbiota transplantation: An emerging trend in cancer patient care. Biochim Biophys Acta Rev Cancer. 2023;1878(6):188990. Williams BA, Mikkelsen D, Flanagan BM, Gidley MJ. Dietary fibre: moving beyond the soluble/insoluble classification for monogastric nutrition, with an emphasis on humans and pigs. J Anim Sci Biotechnol. 2019;10:45. Cantu-Jungles TM, Hamaker BR. New View on Dietary Fiber Selection for Predictable Shifts in Gut Microbiota. mBio. 2020;11(1). Aarnoutse R, Ziemons J, de Vos-Geelen J, Valkenburg-van Iersel L, Wildeboer ACL, Vievermans A, et al. The Role of Intestinal Microbiota in Metastatic Colorectal Cancer Patients Treated With Capecitabine. Clin Colorectal Cancer. 2022;21(2):e87–97. Matsui D. Strategies to Measure and Improve Patient Adherence in Clinical Trials. Pharm Med. 2012;23(5–6):289–97. Additional Declarations Competing interest reported. Declaration of interests: LEH, MAMS, JZ and MLS have received research funding from Danone Research & Innovation. AvH is employed by Danone Research & Innovation, GR was employed by Danone Research & Innovation during the development of the study protocol. MLS has also received funding from Servier and Illumina, all outside the submitted work. JVG has served as a consultant for Amgen, AstraZeneca, MSD, Pierre Fabre, and Servier, and has received institutional research funding from Servier, all outside the submitted work. LVI has served as a consultant for Amgen, MSD, Pierre Fabre, and Servier. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Files Appendix1.Supplementarymaterials.pdf Appendix 1. Supplementary materials Appendix2.Informedconsentmaterial.pdf Appendix 2. Informed consent material Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 06 Apr, 2026 Reviewers agreed at journal 25 Mar, 2026 Reviews received at journal 23 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers invited by journal 16 Mar, 2026 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 23 Dec, 2025 First submitted to journal 23 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8436668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Study protocol","associatedPublications":[],"authors":[{"id":607034935,"identity":"d096282c-51f6-43d2-8644-ad1e7b94b4ec","order_by":0,"name":"Lars E. Hillege","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"Lars","middleName":"E.","lastName":"Hillege","suffix":""},{"id":607034936,"identity":"d03c7a1c-7c23-4614-9721-773079045e4b","order_by":1,"name":"Milou A.M. Stevens","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"Milou","middleName":"A.M.","lastName":"Stevens","suffix":""},{"id":607034937,"identity":"ad83d9e6-3c16-4b8b-885e-5a25a2b0d97f","order_by":2,"name":"Judith de Vos-Geelen","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"Judith","middleName":"","lastName":"de Vos-Geelen","suffix":""},{"id":607034938,"identity":"961d1163-59e2-445d-882d-2e4dccbda35c","order_by":3,"name":"Liselot Valkenburg-van Iersel","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"Liselot","middleName":"Valkenburg-van","lastName":"Iersel","suffix":""},{"id":607034939,"identity":"cd954b0e-be31-42ac-bfd6-8fb33bcebff7","order_by":4,"name":"Sander M.J. van Kuijk","email":"","orcid":"","institution":"Maastricht University Medical Center+","correspondingAuthor":false,"prefix":"","firstName":"Sander","middleName":"M.J. van","lastName":"Kuijk","suffix":""},{"id":607034940,"identity":"7d659bdb-f70c-4828-8b34-b588b29fea31","order_by":5,"name":"Sander S. Rensen","email":"","orcid":"","institution":"Maastricht University Medical Center+","correspondingAuthor":false,"prefix":"","firstName":"Sander","middleName":"S.","lastName":"Rensen","suffix":""},{"id":607034941,"identity":"8745016d-81ad-465d-bd73-ad00595ece46","order_by":6,"name":"Guus Roeselers","email":"","orcid":"","institution":"Danone Research \u0026 Innovation","correspondingAuthor":false,"prefix":"","firstName":"Guus","middleName":"","lastName":"Roeselers","suffix":""},{"id":607034942,"identity":"fd0b03bb-5667-4bbb-b998-83c5fdbe78b4","order_by":7,"name":"Ardy Helvoort","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"Ardy","middleName":"","lastName":"Helvoort","suffix":""},{"id":607034943,"identity":"9574579c-ba32-411a-88bc-0b04bb3eab3e","order_by":8,"name":"John Penders","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Penders","suffix":""},{"id":607034944,"identity":"2431ec11-9e14-4217-9432-87468d96078e","order_by":9,"name":"Marjolein L. Smidt","email":"","orcid":"","institution":"Maastricht University","correspondingAuthor":false,"prefix":"","firstName":"Marjolein","middleName":"L.","lastName":"Smidt","suffix":""},{"id":607034945,"identity":"03a810e1-c7bd-4aa2-87fc-827e71fc4888","order_by":10,"name":"Janine Ziemons","email":"data:image/png;base64,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","orcid":"","institution":"Maastricht University","correspondingAuthor":true,"prefix":"","firstName":"Janine","middleName":"","lastName":"Ziemons","suffix":""}],"badges":[],"createdAt":"2025-12-23 19:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8436668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8436668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104995990,"identity":"71d2e1d0-8f57-4409-ae29-351fb2b62989","added_by":"auto","created_at":"2026-03-19 16:11:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85783,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of the study design.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8436668/v1/f63ecd9ac8f636b33899bb48.jpg"},{"id":104996041,"identity":"fb0a4f51-4fd3-4620-8591-824854b7b79e","added_by":"auto","created_at":"2026-03-19 16:11:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165337,"visible":true,"origin":"","legend":"\u003cp\u003eSchedule of the study period\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e* \u0026nbsp;\u0026nbsp;Most recent CT/MRI prior to start treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e** Routine CT/MRI during 3\u003c/em\u003e\u003csup\u003e\u003cem\u003erd\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e or 4\u003c/em\u003e\u003csup\u003e\u003cem\u003eth\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cycle depending on chemotherapy regimen\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe study period includes 4 cycles of FOLFOX (2 weeks each) or 3 cycles of CAPOX/capecitabine monotherapy (3 weeks each); Fecal samples and questionnaires (T1-T4) are collected during the last 2 days of the last week of the chemotherapy cycle (FOLFOX: week 2, CAPOX/capecitabine monotherapy: week 3); Cycle 4 including associated assessments and T4 fecal samples and questionnaires, are only applicable for patients receiving FOLFOX chemotherapy.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8436668/v1/29af1c2f47bfd4362f17bad2.jpg"},{"id":104996182,"identity":"ab389472-8595-4261-a307-6cc65a274276","added_by":"auto","created_at":"2026-03-19 16:11:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1355761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8436668/v1/022ef932-bf04-43d4-9cf3-fb6054629c8a.pdf"},{"id":104996000,"identity":"6fa0703f-10fd-4f4e-b56c-19a58f4f0ca9","added_by":"auto","created_at":"2026-03-19 16:11:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":74592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAppendix 1. Supplementary materials\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Appendix1.Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8436668/v1/7794ccc5259399627a27207a.pdf"},{"id":104995993,"identity":"4229dd03-3f0d-4840-ac86-fb0d04945888","added_by":"auto","created_at":"2026-03-19 16:11:05","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":148242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAppendix 2. Informed consent material\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Appendix2.Informedconsentmaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8436668/v1/dd2959a8caba95e7adba675f.pdf"}],"financialInterests":"Competing interest reported. Declaration of interests:\nLEH, MAMS, JZ and MLS have received research funding from Danone Research \u0026 Innovation. AvH is employed by Danone Research \u0026 Innovation, GR was employed by Danone Research \u0026 Innovation during the development of the study protocol. MLS has also received funding from Servier and Illumina, all outside the submitted work. JVG has served as a consultant for Amgen, AstraZeneca, MSD, Pierre Fabre, and Servier, and has received institutional research funding from Servier, all outside the submitted work. LVI has served as a consultant for Amgen, MSD, Pierre Fabre, and Servier. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.","formattedTitle":"PREBICC - Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: protocol of a double-blind randomized controlled trial","fulltext":[{"header":"Administrative information","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"639\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eTitle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003ePREBICC: Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: a randomized controlled clinical intervention study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eTrial registration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eOverview of medical research in the Netherlands (OMON)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNL86537.068.24\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eProtocol version\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eVersion 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eFunding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eTKI Agri \u0026amp; Food (LWV20.345)\u003c/p\u003e\n \u003cp\u003eDanone Global Research \u0026amp; Innovation Center\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eAuthor details\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eLars E. Hillege\u003csup\u003e1,2\u003c/sup\u003e, Milou A.M. Stevens\u003csup\u003e1,2\u003c/sup\u003e, Judith de Vos-Geelen\u003csup\u003e1,3\u003c/sup\u003e, Liselot Valkenburg-van Iersel\u003csup\u003e1,3\u003c/sup\u003e, Sander M.J. van Kuijk\u003csup\u003e4\u003c/sup\u003e, Sander S. Rensen\u003csup\u003e2,5\u003c/sup\u003e, Guus Roeselers\u003csup\u003e6\u003c/sup\u003e, Ardy van Helvoort\u003csup\u003e5,6\u003c/sup\u003e, John Penders\u003csup\u003e5,7\u003c/sup\u003e, Marjolein L. Smidt\u003csup\u003e1,2\u003c/sup\u003e, Janine Ziemons\u003csup\u003e1,2\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e1 GROW - Research Institute for Oncology and Reproduction, Maastricht University, Maastricht, the Netherlands\u003c/p\u003e\n \u003cp\u003e2 Department of Surgery, Maastricht University Medical Center+, Maastricht, the Netherlands\u003c/p\u003e\n \u003cp\u003e3 Department of Internal Medicine, Division of Medical Oncology, Maastricht University Medical Center+, Maastricht, the Netherlands\u003c/p\u003e\n \u003cp\u003e4 Department of Clinical Epidemiology and Medical Technology Assessment, Maastricht University Medical Center+, Maastricht, the Netherlands.\u003c/p\u003e\n \u003cp\u003e5 NUTRIM - Institute of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands\u003c/p\u003e\n \u003cp\u003e6 Danone Research \u0026amp; Innovation, Utrecht, the Netherlands\u003c/p\u003e\n \u003cp\u003e7 Department of Medical Microbiology, Infectious Diseases and Infection Prevention, Maastricht University Medical Center+, Maastricht, the Netherlands\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eName and contact information for the trial sponsor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eMaastricht University\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eGROW - Research Institute for Oncology and Reproduction, Maastricht University\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eRole of sponsor and funder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRole of sponsor:\u003c/strong\u003e study design, data collection, lab analysis, data management, data analysis, and interpretation of data, writing of the scientific manuscript, and the decision to submit the manuscript for publication, authority over any of these activities.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRole of funders:\u003c/strong\u003e\u003c/p\u003e\n \u003col\u003e\n \u003cli\u003e\u003cstrong\u003eTKI Agri \u0026amp; Food (LWV20.345):\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDanone Research \u0026amp; Innovation:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003estudy design, lab analysis, and providing feedback on the scientific manuscript.\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eRoles and responsibilities of the research team\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRole and responsibilities p\u003c/strong\u003e\u003cstrong\u003erincipal investigator:\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDesign and conduct of the PREBICC study, preparation of protocol and revisions, completion of electronic CRF [case report forms], publication of study reports, and agreement of final protocol.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRole and responsibilities c\u003c/strong\u003e\u003cstrong\u003eoordinating researcher:\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDesign and conduct of the PREBICC study, preparation of protocol and revisions, preparation of investigators brochure (IB) and electronic CRFs [case report forms], publication of study reports, agreement of final protocol, recruitment of patients and liaising with principle investigator, reviewing progress of study and if necessary agreeing changes to the protocol and/or IB to facilitate the smooth running of the study, study planning, provide annual report to ethics committee, SAEs [Serious adverse events] reporting to ethics committee and in case of study product related SAEs Danone Research \u0026amp; Innovation, responsible for trial master file, budget administration, randomization, and organization of fecal and serum sample collection.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRole and responsibilities s\u003c/strong\u003e\u003cstrong\u003etudy monitor from Clinical Trial Center Maastricht (CTCM):\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eStudy monitoring according to the monitoring plan and data verification.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Introduction ","content":"\u003cp\u003e\u003cstrong\u003eBackground and rationale\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColorectal cancer (CRC) is the third most common cancer in the world (1). In case of (advanced) CRC, the standard systemic treatment is often based on fluoropyrimidines, such as 5-fluorouracil (5-FU). Despite the widespread use of 5-FU-based chemotherapy, there are two significant drawbacks. First, only a limited percentage of patients responds to the treatment. Previous research showed that approximately 35% of patients with advanced CRC responded to capecitabine (an oral prodrug of 5-FU) or 5-FU (2). Second, many patients experience toxicity during 5-FU-based treatment, which might require dose adjustments or even discontinuation of the therapy (3). For instance, a study of 181 patients receiving 5-FU-based treatment for colorectal cancer showed that during the first treatment cycle, 80% of the patients experienced toxicity, mainly mucositis, diarrhea, vomiting, or neutropenia (4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese disadvantages emphasize the relevance of new treatment strategies to optimize efficacy, reduce toxicity, and improve the quality of life (QoL) during 5-FU-based chemotherapy. The gut microbiota represents a promising target to achieve this, since it has been shown to interact with chemotherapeutic drugs and might affect both chemotherapy efficacy and toxicity (5-7). Previous research showed that 5-FU impacts the growth of specific gut bacteria (8-10), which could potentially contribute to a pro-inflammatory microbial milieu and the occurrence of increased (gastrointestinal) toxicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, we recently reported that capecitabine monotherapy reduces fecal levels of the bacteria-derived short-chain fatty acids (SCFA) valerate and caproate (11). Valerate has been shown to cause histone hyperacetylation and growth inhibition in human carcinoma cells and might thus play a role in anti-tumor efficacy (12). Furthermore, baseline fecal levels of the branched-chain fatty acid (BCFA) iso-butyrate were significantly lower in patients with partial response compared to patients with stable or progressive disease, indicating another potential link between the gut microbiota and response to 5-FU-based treatments (11). In line with this, recent in-vitro studies demonstrated that certain bacteria (e.g. \u003cem\u003eE. coli\u003c/em\u003e) are able to directly metabolize 5-FU, with negative consequences for its anti-tumor efficacy (13, 14). The studies of An \u003cem\u003eet al.\u003c/em\u003e, and Yuan \u003cem\u003eet al.\u003c/em\u003e also suggested that 5-FU efficacy is at least partly modulated by intestinal bacteria (15, 16). Recently, a pilot study investigating the effects of fecal microbiota transplantation (FMT) before chemotherapy found that FMT improved the disease control rate, as well as survival, in patients with advanced gastroesophageal cancer (17).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to treatment efficacy and toxicity, cancer cachexia is a factor that significantly influences the patient’s prognosis and quality of life. Different associations between the intestinal microbiota and cancer cachexia have been described (as reviewed in (18)). In a previous clinical study, we reported altered abundance of specific taxa and reduced levels of the SCFA acetate in cachectic cancer patients compared to non-cachectic cancer patients and healthy controls (19). Furthermore, cachexia-associated alterations in intestinal microbiota composition can be found, commonly characterized by an overgrowth of taxa belonging to the pro-inflammatory Enterobacteriaceae family (20-22).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the studies described above, it can be concluded that targeted modulation of the intestinal microbiota has the potential to optimize 5-FU-based chemotherapy by increasing therapy efficiency, decreasing toxicity, and reducing cancer cachexia. In this context, the principal aim would be to stimulate the growth of gut bacteria with beneficial properties, thereby counteracting the overgrowth of bacteria with pro-inflammatory properties and/or an unwanted role in 5-FU metabolism. To achieve this, prebiotics are a promising strategy.Prebiotics are substrates that are selectively fermented by microorganisms and confer a health benefit to the host, by stimulating the growth and/or activity of beneficial bacteria (23). In addition, prebiotic fermentation leads to the formation of SCFA, which not only modulate the growth of cancer cells (as described above), but also have potent anti-inflammatory as well as beneficial metabolic effects (24-26). Supplementation with the prebiotic fructo-oligosaccharide (FOS) in a mouse model of 5-FU-induced mucositis improved gut integrity by enhancing SCFA production, maintaining tight junctions, and reducing inflammation (27). In previous in-vitro experiments, we found that prebiotic fiber mixtures also exert these positive effects in the presence of 5-FU (28). As a next step, the PREBICC study aims to investigate whether these beneficial effects can also be achieved in CRC patients during 5-FU-based chemotherapy and whether these microbiota alterations improve 5-FU toxicity and/or efficacy as well as cachexia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aims to investigate the effects of daily administration of a prebiotic fiber mixture in a nutritionally complete Oral Nutritional Supplement (ONS) on the abundance of microbial taxa with beneficial properties (e.g. \u003cem\u003eBifidobacterium\u003c/em\u003e), as well as on intestinal microbiota composition and diversity, compared to a control ONS, in patients with advanced CRC treated with 5-FU-based chemotherapy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs exploratory objectives, this study will assess (1) thefeasibility, tolerance, and compliance of a nutritional intervention with a prebiotic ONS in patients with advanced CRC, (2) the effects of the prebiotic ONS on fecal and blood parameters, tumor response, chemotherapy toxicity as well as the metabolic phenotype (e.g. body weight, body composition, inflammatory profile), and (3) the relationship with the patient-reported QoL and physical functioning.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis intervention study is a double-blind randomized controlled trial in patients with advanced CRC receiving 5-FU-based chemotherapy and will be performed in the Maastricht University Medical Center (MUMC+), the Netherlands, with the potential for future expansion to a multicenter setting.\u0026nbsp;Patients will be stratified based on their assigned chemotherapy regimen (FOLFOX, CAPOX, or capecitabine monotherapy) before 1:1 randomization. The block size will be either 2 or 4.\u0026nbsp;Patients will be randomized to either the test group (ONS enriched with prebiotic fibers) or the control group (isocaloric, isonitrogenous ONS without prebiotic fibers). Patients will start with the prebiotic or control ONS at least three days prior to the start of the 5-FU-based treatment. They will continue to take it twice daily for the duration of either four cycles of FOLFOX (in total 8 weeks) or three cycles of CAPOX or capecitabine monotherapy (in total 9 weeks).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFecal samples will be collected at multiple time points throughout the study period: before the start of the intervention (T0) and in the last two days of chemotherapy cycles 1 (T1), 2 (T2), 3 (T3), and 4 (T4, for FOLFOX only). Simultaneously, patients will fill in questionnaires concerning amongst others body weight and length, experienced side effects of chemotherapy (according to the Common Terminology Criteria for Adverse Events (CTCAE)), gastrointestinal tolerance and stool consistency (Bristol stool scale), QoL (EORTC QLQ-C30 (29) and EQ-5D-5L (30)), current nutritional status (Patient-Generated Subjective Global Assessment (PG-SGA) score), and physical performance (Karnofsky Performance Score (KPS)). A food habits questionnaire (FQ18) will be completed at the start of the study period (T0) (31). In addition, repeated blood samples will be collected to analyze amino acids, C-reactive protein (CRP), and SCFA concentrations. Changes in dietary habits, compliance, and changes in patients’ body weight will be recorded throughout the study period. Routine CT/MRI scans before and during the third or fourth chemotherapy cycle will be used to evaluate body composition (CT scans only), as well as tumor response (based on the ‘Response Evaluation Criteria in Solid Tumors’ (RECIST) (32)) (Figure 1).\u0026nbsp;\u003c/p\u003e"},{"header":"Methods: Participants, interventions, and outcomes","content":"\u003cp\u003e\u003cstrong\u003eEligibility criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study population includes patients diagnosed with advanced CRC who are scheduled for treatment with 5-FU-based therapy (FOLFOX, CAPOX, or capecitabine monotherapy). Simultaneous treatment with bevacizumab, panitumumab, or cetuximab is permitted, if no systemic antibiotics are used (topical antibiotics are allowed). A full list of all the inclusion and exclusion criteria is provided in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Inclusion and exclusion criteria\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInclusion\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecriteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cul\u003e\n \u003cli\u003ePatients diagnosed with advanced CRC who will be treated with 5-FU-based therapy (FOLFOX, CAPOX, capecitabine monotherapy)\u003c/li\u003e\n \u003cli\u003eSimultaneous treatment with bevacizumab, panitumumab, or cetuximab is allowed, provided that no systemic antibiotics are used (topical antibiotics are allowed)\u003c/li\u003e\n \u003cli\u003eProficient use of the Dutch language\u003c/li\u003e\n \u003cli\u003eThe patient is older than 18 years of age\u003c/li\u003e\n \u003cli\u003ePerformance (ECOG/WHO) score 0-2 (33)\u003c/li\u003e\n \u003cli\u003eWritten informed consent\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eExclusion criteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cul\u003e\n \u003cli\u003eMicrosatellite instability (MSI) or deficient mismatch repair (MMR) proteins\u003c/li\u003e\n \u003cli\u003eKnown abnormal DPYD variants (single nucleotide polymorphisms) and/or reduced dihydropyrimidine dehydrogenase (DPD) enzyme function\u003c/li\u003e\n \u003cli\u003ePresence of ileostomy\u003c/li\u003e\n \u003cli\u003ePregnant or nursing\u003c/li\u003e\n \u003cli\u003ePrevious systemic therapy for advanced CRC. If the patient received prior (neo)adjuvant systemic therapy, it must have been completed at least 6 months before the diagnosis of the advanced disease\u003c/li\u003e\n \u003cli\u003eTherapeutic systemic antibiotics used less than four weeks before the start of 5-FU-based therapy (topical antibiotics are allowed)\u003c/li\u003e\n \u003cli\u003eAbdominal radiotherapy less than two weeks before the start of the 5-FU-based therapy\u003c/li\u003e\n \u003cli\u003eUse of pro- and/or prebiotics during the study period\u003c/li\u003e\n \u003cli\u003eInflammatory bowel disease (Crohn\u0026rsquo;s disease or ulcerative colitis)\u003c/li\u003e\n \u003cli\u003eSimultaneous treatment with irinotecan chemotherapy\u003c/li\u003e\n \u003cli\u003eKnown allergy to any ingredients present in the test or control ONS, requiring a fiber-free diet, or suffering galactosemia or lactose intolerance\u003c/li\u003e\n \u003cli\u003eSimultaneous participation in another medical-scientific intervention study\u003c/li\u003e\n \u003cli\u003ePhysically or mentally incapable or incompetent\u003c/li\u003e\n \u003cli\u003eAll conditions that, in the opinion of the physician, are not suitable for participation in this study (e.g. severe renal failure).\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eIntervention\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDescription\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intervention for this study involves twice daily consumption of an ONS with or without the addition of prebiotic fibers for eight or nine weeks depending on the chemotherapy regimen. Patients will start the ONS intake at least three days prior to the start of 5-FU-based chemotherapy. The test and control ONS are nutritionally complete, high-energy (300 kcal), high-protein (12g), ready-to-drink supplements, intended for medical use (Nutricia, Zoetermeer, The Netherlands). The test ONS additionally contains 4.5g of fibers, a mixture of galacto-oligosaccharides (GOS), FOS, and pectin per bottle. Both the test and control ONS are supplied in blinded 125 mL bottles and will be available in two flavors: vanilla and strawberry (Appendix 1, supplementary table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eModifications\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent modifications to the study procedure are possible. First of all, patients are free to withdraw from the study at any time and without any consequences. Secondly, the intervention period might be prolonged if the medical oncologist adapts the 5-FU treatment schedule of a patient. If the aggregated total treatment delay throughout the study period exceeds four weeks, study participation of this patient will be discontinued after the collection of a final fecal sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThirdly, if a patient requires additional calories to maintain weight during chemotherapy, the dosage of the test/control ONS will be increased to meet the energy requirements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, in case of non-compliance, the researcher can discontinue participation of a patient in the trial. If a patient experiences study product-related side effects that cause significant harm or make continued participation impractical, the researcher may choose to withdraw the patient from the study, especially in the case of a serious adverse event (SAE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdherence\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients are regularly interviewed concerning their consuming experiences and should return any unconsumed drinks at the visits with the researcher at the start of each cycle during the study period. This information will be used to monitor compliance. To increase adherence, patients can choose one or a combination of the flavors: vanilla or strawberry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMain outcomes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main outcomes include the differences in shifts (∆baseline vs end of study) of the relative abundance of specific microbial taxa (e.g.\u0026nbsp;\u003cem\u003eBifidobacterium\u003c/em\u003e), as well as of overall intestinal microbiota composition and diversity between test and control group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExploratory outcomes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLongitudinal changes in:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParameters assessed in feces\u003c/em\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eIntestinal microbiome composition parameters: e.g., alpha \u0026amp; beta diversity and taxonomic and functional composition during all timepoints\u003c/li\u003e\n \u003cli\u003eFunctional capacity of the intestinal microbiota during all timepoints\u003c/li\u003e\n \u003cli\u003eLevels of SCFA and BCFA\u003c/li\u003e\n \u003cli\u003epH and dry weight\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eConcentrations of measures of gut inflammation (calprotectin), immune status and gut integrity (secretory immunoglobulin A (sIgA)) and microbial metabolism (ammonia) (34-36)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cem\u003eParameters assessed in blood\u003c/em\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eAmino acids concentrations\u003c/li\u003e\n \u003cli\u003eC-reactive protein (CRP) concentrations\u003c/li\u003e\n \u003cli\u003eLevels of SCFA\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cem\u003eClinical parameters\u003c/em\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eNutritional parameters: body weight, BMI, and PG-SGA score (37)\u003c/li\u003e\n \u003cli\u003eDietary habits: FQ18 questionnaire and follow-up questionnaires during the study (31)\u003c/li\u003e\n \u003cli\u003eExperienced toxicity related to chemotherapy: CTCAE questionnaire (38)\u003c/li\u003e\n \u003cli\u003ePhysical performance: Karnofsky performance score (39)\u003c/li\u003e\n \u003cli\u003eQuality of life: EORTC QLQ-C30 and EQ-5D-5L (29, 30)\u003c/li\u003e\n \u003cli\u003eBody composition: measured using routine CT scan at L3 level\u003c/li\u003e\n \u003cli\u003eTumor response: RECIST criteria (32)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cem\u003eTolerance parameters\u003c/em\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003ePrebiotic and control ONS compliance\u003c/li\u003e\n \u003cli\u003eGastrointestinal tolerance: Bristol stool scale and diarrhea prevalence (40)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOther outcomes\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the main and secondary outcomes, the following patient characteristics will be recorded: age, sex, pre-study weight loss in the last six months, and medication and supplement usage before and during the study. In addition, information concerning medical history and hematological parameters, i.e. leukocytes, neutrophils, and thrombocytes, will be retrieved from medical records.\u003c/p\u003e\n\u003cp\u003eAdditional fecal and blood samples will be stored to enable potential future analysis of other exploratory outcomes, for instance fecal levels of tryptophan (metabolites) and D-Amino acids in feces or blood cytokine levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipant timeline\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn overview of the study timeline is depicted in Figure 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main outcome of this study is the relative abundance of members of the genus\u0026nbsp;\u003cem\u003eBifidobacterium\u003c/em\u003e (log10 copies/gram wet weight feces) in fecal samples, measured at baseline (pre-5-FU-based chemotherapy and pre-ONS-intervention) and post-intervention between the test and control group. This primary endpoint involves comparing the mean change from baseline (post-baseline minus baseline) between test and control group. The difference between groups will be evaluated using the independent-samples t-test or linear regression models.\u003c/p\u003e\n\u003cp\u003eThe sample size calculation is based on estimates from a prior study using a similar prebiotic ONS (OMON registration\u0026nbsp;number\u0026nbsp;NL1693). Based on this study, a standard deviation of 0.35 is assumed. The smallest detectable difference with sufficient power is set to 0.34. Based on the provided estimates and parameters, a sample size of 46 patients, with 23 in each group (test and control), is considered sufficient to detect a statistically significant difference in Bifidobacteria changes between the test and control groups using a two-sided independent-samples t-test, when testing with a significance level (\u0026alpha;) of 0.05, and a power of 90%.\u003c/p\u003e\n\u003cp\u003eConsidering an anticipated dropout rate of 25% for the intervention period, the total number of subjects to be enrolled is adjusted to 62, with 31 patients in each of the two groups (intervention and control). This adjustment ensures that even with the expected dropouts, there will be a sufficient number of patients to maintain the statistical power of the study as planned.\u003c/p\u003e\n\u003cp\u003eThe sample size calculation was performed using the formula from Chow\u0026nbsp;\u003cem\u003eet al\u003c/em\u003e. with R software for statistical computing (41).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruitment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedical oncologists (in training) at the MUMC+ will ask eligible patients for their consent to be contacted by the researcher and/or will briefly inform the patient about the study. Subsequently, the researcher will further inform the patient about the aims of the study, explain the sampling procedure, and expectations throughout the intervention. A patient information letter, a flyer containing all essential information about the study, and informed consent form will be provided. After a reflection time of at least 24 hours up until 7 days when treatment schedule and anticipated start of treatment allows, patients are asked if they would like to participate. If the patient agrees to proceed, an appointment with the researcher is scheduled and informed consent (Appendix 2) will be signed together.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: Assignment of interventions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAllocation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEligible patients will be allocated to one of two groups: test or control group. To ensure balance across these two groups regarding key prognostic variables, patients will be stratified based on their assigned chemotherapy regimen prior to randomization (FOLFOX, CAPOX, capecitabine monotherapy). Following stratification, patients will undergo a 1:1 randomization with block sizes of either 2 or 4 to ensure an equal number of patients in each group. The randomization process will be facilitated using Castor and ALEA systems. ALEA will automatically assign each patient to either the test or control group, adhering to a predefined block randomization scheme. Following the assignment, ALEA selects a product number from a predetermined list, which is managed by an unblinded researcher in collaboration with the supplies manager. This list comprises product numbers uniquely associated with either the prebiotic or control ONS, ensuring that patients receive the correct ONS according to their group assignment. This ensures that blinded researchers, even after an unblinding event, will not be able to ascertain the group assignments of other patients within the study. The study coordinator will perform the generation of the allocation sequence, the enrollment of patients, and the assignment of patients to interventions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlinding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeither the patients nor the researchers know which ONS is administered until the clinical trial concludes. Only one independent researcher and the supplies manager, who are not involved in patient contact, sample processing, or data analysis, will be unblinded. Emergency unblinding of the allocation will occur if patients experience SAEs which are likely to be related to the study product.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: Data collection, management, and analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection plan\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFecal samples\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFecal samples will be collected at several time points throughout the study period.\u0026nbsp;Patients are asked to collect the fecal samples at home in preservation-free tubes (Sarstedt) and to immediately store them in the freezer. The samples will be transported to the hospital in a cooled container (Sarstedt) to prevent thawing and will be stored at -80\u0026nbsp;\u0026deg;C. Metagenomic sequencing will be performed to quantify fecal microbiota composition, diversity, functional capacity, and taxonomic abundances. To this end, total DNA will be extracted from fecal samples using the QIAmp DNA Stool Mini Kit (Qiagen) where the manufacturer\u0026rsquo;s protocol will be followed but with an addition of two bead-beating steps. The Illumina Nextera XT DNA Library Prep kit (Illumina, San Diego, USA) will be used as per the manufacturer\u0026rsquo;s instructions. Library quality control will be checked, and the samples will be loaded onto the NovaSeq 6000 platform. Subsequently, metabolic activity of the gut microbiota will be assessed. The pH, SCFA levels (i.e. acetate, propionate, butyrate, valerate), BCFA levels (iso-butyrate, iso-valerate), ammonia, and calprotectin concentrations will be measured. Details on the methods that will be used to measure these physiological parameters have been described previously (42, 43). For the assessment of sample dry weight, 500mg of frozen feces will be dried in a vacuum dryer (Eppendorf) for five hours and will be weighted afterwards.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlood samples\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples will be collected, if possible together with routine blood samples, before every cycle during the intervention period. The concentrations of amino acids, CRP, and levels of SCFA will be measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuestionnaires\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the intervention period, patients will complete the following questionnaires.\u0026nbsp;\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u003cem\u003ePatient-Generated Subjective Global Assessment (PG-SGA)\u0026nbsp;\u003c/em\u003eis a patient-reported questionnaire for assessment of the nutritional status in cancer patients (37). In the current study, the Short Form version will be used to assess the current nutritional status at the same time as fecal sample collection.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eFQ18\u0026nbsp;\u003c/em\u003eis a Dutch web-based food habits questionnaire developed by Wageningen University \u0026amp; Research. It assesses intake of energy, macronutrients, type of fatty acids and dietary fiber (31). The FQ18 will be completed only once at the beginning of the study period.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eCommon Terminology Criteria for Adverse Events (CTCAE)\u0026nbsp;\u003c/em\u003ewill be used to assess chemotherapy side effects during each cycle in the study period, e.g. nausea and diarrhea (38).\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eKarnofsky Performance Score (KPS)\u003c/em\u003e will be used to assessphysical performance as this validated questionnaire assesses physical and psychosocial difficulties (39).\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eEORTC QLQ-C30\u0026nbsp;\u003c/em\u003eis widely used to assess the QoL in cancer patients, including those with colorectal cancer. Developed by the European Organisation for Research and Treatment of Cancer (EORTC), it measures various aspects of QoL, such as physical, emotional, cognitive, and social functioning, as well as symptom burden. For colorectal cancer patients, relevant items may include issues related to eating, fatigue, pain, and gastrointestinal symptoms (29).\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eEQ-5D-5L\u0026nbsp;\u003c/em\u003eis a standardized measure of health-related QoL, assessing five dimensions of health with five severity levels each (30).\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eBristol Stool Scale\u0026nbsp;\u003c/em\u003ewas developed in 1997 for the purpose of evaluating intestinal transit time.Utilization of this scale enables the assessment of gastrointestinal tolerance, as alterations in bowel habits resultant from the intervention can be monitored (40).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBody composition and tumor response\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT or MRI scans will be made for routine care before the intervention and during the third or fourth treatment cycle. These CT scans will be used to evaluate body composition (based on the image at the third lumbar vertebra (L3) level) and CT or MRI scans will be used to evaluate tumor response (based on the RECIST criteria (32)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdditional clinical information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical information from the questionnaires will be completed with information derived from the patient\u0026rsquo;s medical record, for instance concerning toxicity, dose adjustments, medical history, and co-medication. In addition, the blood parameters, as well as parameters for liver and kidney function will be assessed in the context of standard care and retrieved from the patient\u0026rsquo;s medical record.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFollow up after participation withdraw\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen a patient decides to withdraw from the study, any unused study products and other study materials will be returned in consultation with the researcher. Approximately two weeks after taking the last study product, patients will be contacted to discuss any delayed side effects and to evaluate their participation in the intervention study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePersonal data will be handled with strict care securing the patients\u0026apos; privacy according to the EU General Data Protection Regulation. All patients and their samples receive a pseudonymized study code. The key linking study code to patient data (hospital ID and name) will only be accessible to the primary researcher as well as the (coordinating) researcher(s).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElectronic data will be safely stored and backed up in an electronic data capture system. The research process will be documented. Data validation messages are implemented to provide warnings for potential errors in the data input. Additional quality checks will be performed before, during and after the data collection. All study samples will be centrally stored at the Biobank of the MUMC+ until used for analysis. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses will be performed with R and R Studio. All analyses will be performed according to the intention to treat principle. In case of missing data in over 5% of patient records, an imputation method will be chosen depending on the extent of missing data and the likely missing data mechanism(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMain outcomes\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data from metagenomic sequencing will be processed further and analyzed using state-of-the-art in-house data analysis pipelines. The main study parameter is focused on exploring differences in the relative abundance of specific bacterial taxa (∆baseline vs end of study) between the test group and control group. For this purpose, differential abundance analyses, which were specifically developed for compositional data (CoDA) will be used. Furthermore, generalized linear mixed-effects models on centered log ratio (CLR) transformed microbial counts will be used for repeated measurements. Overall intestinal microbiota composition (\u0026beta;-diversity) and clustering of samples will be visualized by means of Principal Component Analysis (PCA). The Shannon index and observed species richness will be calculated as measures of microbial diversity (\u0026alpha;-diversity). Intention-to-treat analyses will be performed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSecondary outcomes\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether parametric or nonparametric tests should be performed, normality will be assessed for all variables based on histograms, Q-Q-Plots, and the Shapiro-Wilk test. Longitudinal analysis (changes during the study period) will be conducted using Friedman\u0026rsquo;s ANOVA or linear mixed-effects models. For cross-sectional analyses comparing the intervention and control group at specific time points, the Kruskal\u0026ndash;Wallis or Mann\u0026ndash;Whitney U test will be used for non-normally distributed data, and the t-test or ANOVA if the assumption of normality is met. To analyse associations between different clinical variables of interest, Spearman correlation will be computed, and p-values will be adjusted for multiple testing by means of False Discovery Rate (FDR) adjustments according to the Benjamini and Hochberg procedure (44). Permutational multivariate analysis of variance (PERMANOVA) will be performed to assess the correlation between microbial profiles and (clinical) determinants as it is a non-parametric method and it accommodates the complex nature of microbial community data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: Monitoring\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study will be monitored by the Clinical Trial Center Maastricht (CTCM), according to a predefined monitoring plan. The monitor will be independent from the sponsor of this study. There will be no Data and Safety Monitoring Board (DSMB) installed, since the product under investigation has a history of safe use and is not expected to cause severe undesired side effects. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHarms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll adverse events (AE) that occur during the study period will be recorded,\u0026nbsp;whether or not considered related to the intervention. Furthermore, the researcher will report SAEs to the sponsor, directly after obtaining knowledge of the events, except for the following SAEs that occur commonly in the patient population under investigation:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eElective hospitalization or hospitalization related to technical, practical, or social reasons (i.e. prolonging hospital stay to arrange aftercare)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHospitalization due to common side effects of the chemotherapeutic regimen assessed by the treating physician\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEvents as a result of the progression of the disease under study.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eSAEs will be reported through the web portal ToetsingOnline to the METC azM/UM. In case of SAEs considered to be related to the use of the study product, the supplier Danone Research \u0026amp; Innovation will be informed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp\u003eFluoropyrimidine-based treatments, such as 5-FU and capecitabine, either in combination with oxaliplatin or irinotecan, form the backbone of advanced CRC therapy (45), although only part of the patients respond well (2). Additionally, many patients experience significant toxicity, necessitating dose adjustments or discontinuation, and further decreasing 5-FU efficacy (3). These limitations highlight the need for new strategies to optimize efficacy, reduce toxicity, and improve QoL during 5-FU-based chemotherapy. Since the gut microbiota has been shown to interact with 5-FU and might impact its efficacy and toxicity, the PREBICC study aims to investigate whether an ONS enriched with prebiotic fibers could stimulate abundance of bacteria with beneficial properties and modulate gut microbiota composition and diversity in patients with advanced CRC treated with 5-FU-based chemotherapy. Furthermore, the study aims to explore whether microbiota alterations correlate with improvements in 5-FU toxicity, tumor response, physical functioning, metabolic phenotype, and QoL, as well as evaluates the feasibility of such an intervention.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile there are also other on-going studies using prebiotics in cancer patients (46, 47), most trials currently focus on probiotics or FMT (48). We decided to test prebiotics, because they do not contain living bacteria, are considered to induce more holistic microbiota changes compared to probiotics, and are less invasive compared to FMT. Since different prebiotic fibers might exert distinct effects depending on their physicochemical properties (49, 50), we chose the prebiotic ONS based on our previous in-vitro experiments (28). Furthermore, an additional strength of this clinical trial is its randomized double-blinded design.\u003c/p\u003e\n\u003cp\u003eThe potential implications of this study are substantial. If daily administration of a prebiotic ONS proves effective in modulating the gut microbiota, enhancing chemotherapy efficacy, and reducing toxicity, it could lead to significant improvements in the standard treatment protocols for advanced CRC. By optimizing gut microbiome composition, patients may experience fewer side effects, allowing for more consistent dosing and potentially better overall outcomes. Furthermore, a reduction in cancer cachexia could greatly improve patients' QoL and prognosis. Additionally, PREBICC will provide important insights into the feasibility of gut microbiota modulation during chemotherapy and the specific needs of patients with advanced cancer. Altogether, these results will greatly contribute to the tailor-made and personalized design of prebiotic interventions and implementation into clinical practice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are several limitations to the current study. First, we previously reported considerable inter-individual variability in gut microbiota composition among CRC patients treated with capecitabine, most likely due to diverse medical histories and the administration of antibiotics and other co-medication (51). We are unable to exclude that this will also occur in the PREBICC cohort, which could complicate the detection of 5-FU or prebiotic-induced effects. Secondly, the efficacy of the intervention mainly depends on compliance of the patients (52). Reliable assessment of patient compliance is challenging, and the methods used in this study can only estimate compliance. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the PREBICC study protocol outlines a novel approach to enhance the efficacy and reduce the toxicity of 5-FU-based chemotherapy in patients with advanced CRC through the modulation of the gut microbiota using prebiotics. This exploratory research holds the potential to significantly impact clinical practices and improve patient outcomes, paving the way for future studies and advancements in microbiome-based therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eAbbreviation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eDefinition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e5-FU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e5-fluorouracil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eAE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eAdverse events\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eBCFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eBranched-chain fatty acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCCMO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eCentrale Commissie Mensgebonden Onderzoek\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCLR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eCentered log ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCoDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eCompositional data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCRC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eColorectal cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCRFs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eCase report forms\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eC-reactive protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCTCAE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eCommon Terminology Criteria for Adverse Events\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eCTCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eClinical Trial Center Maastricht\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eDPDD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eDihydropyrimidine dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eDSMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eData and Safety Monitoring Board\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eFDR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eFalse Discovery Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eFMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eFecal microbiota transplantation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eFOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eFructo-oligosaccharide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eGOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eGalacto-oligosaccharides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eInvestigators brochure\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eICH-GCP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eInternational Conference on Harmonization Good Clinical Practice\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eICMJE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eInternational Committee of Medical Journal Editors\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eKPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eKarnofsky Performance Score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eMMR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eMismatch repair\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eMSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eMicrosatellite instabillity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eMUMC+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eMaastricht University Medical Center+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eOMON\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eOverview of medical research in the Netherlands\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eONS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eOral nutritional supplement\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003ePCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003ePrincipal Component Analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003ePERMANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003ePermutational multivariate analysis of variance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003ePG-SGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003ePatient-Generated Subjective Global Assessment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eQoL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eQuality of life\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eRECIST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eResponse Evaluation Criteria in Solid Tumors\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eSAE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eSerious adverse event\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eSCFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eShort-chain fatty acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003esIgA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eSecretory immunoglobulin A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics and dissemination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethical Committee azM/UM (NL86537.068.24 / METC24-011) and Institutional Review Board of Maastricht UMC+. The study will be conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki and consistent with the International Conference on Harmonization Good Clinical Practice (ICH-GCP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtocol amendments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny essential changes in the study protocol and associated documents will be submitted to the medical ethical committee for assessment. If changes need to be implemented, this will be communicated to the parties involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients will be informed in detail about the study aims and procedures. After a reflection time, patients are asked whether they would like to participate or not and informed consent will be signed in the presence of the researcher.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLEH, MAMS, JZ and MLS have received research funding from Danone Research \u0026amp; Innovation. AvH is employed by Danone Research \u0026amp; Innovation, GR was employed by Danone Research \u0026amp; Innovation during the development of the study protocol. MLS has also received funding from Servier and Illumina, all outside the submitted work. JVG has served as a consultant for Amgen, AstraZeneca, MSD, Pierre Fabre, and Servier, and has received institutional research funding from Servier, all outside the submitted work. LVI has served as a consultant for Amgen, MSD, Pierre Fabre, and Servier. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData access\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data handling procedures will adhere to the regulations outlined in the EU General Data Protection Regulation and the Dutch Act on Implementation of the General Data Protection Regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by TKI Agri \u0026amp; Food (LWV20.345) and Danone Global Research \u0026amp; Innovation Center. The funding bodies had no role in the study design, data collection, analysis, or interpretation of the data, nor in the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissemination policy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe principal investigator will be responsible for the public disclosure/publication of the study outcome. Scientific findings will be published in open-access, peer-reviewed journals, adhering to the “Centrale Commissie Mensgebonden Onderzoek” (CCMO) guideline and the International Committee of Medical Journal Editors (ICMJE) criteria for co-authorship. Additionally, results will be disseminated at various forums, including stakeholder-specific conferences, symposia, workshops, and training events. Other platforms for sharing information will include social media, press releases, project websites, newsletters, and science weeks. Data used and/or analyzed will be available after completion of the study from the corresponding author on reasonable request. Raw sequencing data will be submitted to an online repository. Furthermore, the trial is registered in a public trial registry (OMON) and sequencing data obtained in the context of this study will be deposited in a public repository after the completion of the analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAu\u003c/strong\u003e\u003cstrong\u003ethor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLEH: Conceptualization, Methodology, Funding Acquisition, Project Administration, Investigation, Resources, Writing - Original Draft, Writing - Review \u0026amp; Editing, Ethics Approval, Data Curation, Validation, Visualization. MAMS: Conceptualization, Methodology, Project Administration, Investigation, Resources, Writing - Original Draft, Writing - Review \u0026amp; Editing, Ethics Approval, Data Curation, Validation, Visualization. JVG: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review \u0026amp; Editing, Data Curation, Validation. LVI: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review \u0026amp; Editing, Data Curation, Validation. SMJK: Conceptualization, Methodology, Writing - Review \u0026amp; Editing, Validation. SSR: Conceptualization, Funding Acquisition, Writing - Review \u0026amp; Editing. GR: Conceptualization, Funding Acquisition, Writing - Review \u0026amp; Editing. AH: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review \u0026amp; Editing, Validation. JP: Conceptualization, Methodology, Funding Acquisition, Resources, Supervision, Writing - Review \u0026amp; Editing, Validation. MLS: Conceptualization, Funding Acquisition, Investigation, Resources, Supervision, Writing - Review \u0026amp; Editing, Data Curation, Validation. JZ: Conceptualization, Methodology, Funding Acquisition, Project Administration, Investigation, Resources, Supervision, Writing - Original Draft, Writing - Review \u0026amp; Editing, Ethics Approval, Data Curation, Validation, Visualization.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRawla P, Sunkara T, Barsouk A. Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors. Prz Gastroenterol. 2019;14(2):89\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrelli F, Cabiddu M, Barni S. 5-Fluorouracil or capecitabine in the treatment of advanced colorectal cancer: a pooled-analysis of randomized trials. Med Oncol. 2012;29(2):1020\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTimmers L, Boons CC, Mangnus D, Van de Ven PM, Van den Berg PH, Beeker A, et al. Adherence and Patients' Experiences with the Use of Capecitabine in Daily Practice. Front Pharmacol. 2016;7:310.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGusella M, Crepaldi G, Barile C, Bononi A, Menon D, Toso S, et al. Pharmacokinetic and demographic markers of 5-fluorouracil toxicity in 181 patients on adjuvant therapy for colorectal cancer. Ann Oncol. 2006;17(11):1656\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexander JL, Wilson ID, Teare J, Marchesi JR, Nicholson JK, Kinross JM. Gut microbiota modulation of chemotherapy efficacy and toxicity. Nat Rev Gastroenterol Hepatol. 2017;14(6):356\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAarnoutse R, Ziemons J, Penders J, Rensen SS, de Vos-Geelen J, Smidt ML. The Clinical Link between Human Intestinal Microbiota and Systemic Cancer Therapy. Int J Mol Sci. 2019;20(17).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong SH, Yu J. Gut microbiota in colorectal cancer: mechanisms of action and clinical applications. Nat Rev Gastroenterol Hepatol. 2019;16(11):690\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaier L, Pruteanu M, Kuhn M, Zeller G, Telzerow A, Anderson EE, et al. Extensive impact of non-antibiotic drugs on human gut bacteria. Nature. 2018;555(7698):623\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSougiannis AT, VanderVeen BN, Enos RT, Velazquez KT, Bader JE, Carson M, et al. Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota. Brain Behav Immun. 2019;80:44\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvalho R, Vaz A, Pereira FL, Dorella F, Aguiar E, Chatel JM, et al. Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP. Sci Rep. 2018;8(1):15072.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiemons J, Aarnoutse R, Heuft A, Hillege L, Waelen J, de Vos-Geelen J et al. Fecal levels of SCFA and BCFA during capecitabine in patients with metastatic or unresectable colorectal cancer. Clin Exp Med. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHinnebusch BF, Meng S, Wu JT, Archer SY, Hodin RA. The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. J Nutr. 2002;132(5):1012\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaCourse KD, Zepeda-Rivera M, Kempchinsky AG, Baryiames A, Minot SS, Johnston CD, et al. The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota. Cell Rep. 2022;41(7):111625.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpanogiannopoulos P, Kyaw TS, Guthrie BGH, Bradley PH, Lee JV, Melamed J, et al. Host and gut bacteria share metabolic pathways for anti-cancer drug metabolism. Nat Microbiol. 2022;7(10):1605\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn J, Ha EM. Combination Therapy of Lactobacillus plantarum Supernatant and 5-Fluouracil Increases Chemosensitivity in Colorectal Cancer Cells. J Microbiol Biotechnol. 2016;26(8):1490\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan L, Zhang S, Li H, Yang F, Mushtaq N, Ullah S, et al. The influence of gut microbiota dysbiosis to the efficacy of 5-Fluorouracil treatment on colorectal cancer. Biomed Pharmacother. 2018;108:184\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Clercq NC, van den Ende T, Prodan A, Hemke R, Davids M, Pedersen HK, et al. Fecal Microbiota Transplantation from Overweight or Obese Donors in Cachectic Patients with Advanced Gastroesophageal Cancer: A Randomized, Double-blind, Placebo-Controlled, Phase II Study. Clin Cancer Res. 2021;27(13):3784\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiemons J, Smidt ML, Damink SO, Rensen SS. Gut microbiota and metabolic aspects of cancer cachexia. Best Pract Res Clin Endocrinol Metab. 2021:101508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUbachs J, Ziemons J, Soons Z, Aarnoutse R, van Dijk DPJ, Penders J, et al. Gut microbiota and short-chain fatty acid alterations in cachectic cancer patients. J Cachexia Sarcopenia Muscle. 2021;12(6):2007\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBindels LB, Beck R, Schakman O, Martin JC, De Backer F, Sohet FM, et al. Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model. PLoS ONE. 2012;7(6):e37971.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBindels LB, Neyrinck AM, Claus SP, Le Roy CI, Grangette C, Pot B, et al. Synbiotic approach restores intestinal homeostasis and prolongs survival in leukaemic mice with cachexia. Isme j. 2016;10(6):1456\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026ouml;tgens SA, Brossel H, Sboarina M, Catry E, Cani PD, Neyrinck AM, et al. Klebsiella oxytoca expands in cancer cachexia and acts as a gut pathobiont contributing to intestinal dysfunction. Sci Rep. 2018;8(1):12321.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibson GR, Hutkins R, Sanders ME, Prescott SL, Reimer RA, Salminen SJ, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol. 2017;14(8):491\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Beek CM, Dejong CHC, Troost FJ, Masclee AAM, Lenaerts K. Role of short-chain fatty acids in colonic inflammation, carcinogenesis, and mucosal protection and healing. Nutr Rev. 2017;75(4):286\u0026ndash;305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity. 2014;40(1):128\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrison DJ, Preston T. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes. 2016;7(3):189\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvalho PLA, Andrade MER, Trindade LM, Leoc\u0026aacute;dio PCL, Alvarez-Leite JI, Dos Reis DC, et al. Prophylactic and therapeutic supplementation using fructo-oligosaccharide improves the intestinal homeostasis after mucositis induced by 5- fluorouracil. Biomed Pharmacother. 2021;133:111012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiemons J, Hillege LE, Aarnoutse R, de Vos-Geelen J, Valkenburg-van Iersel L, Mastenbroek J, et al. Prebiotic fibre mixtures counteract the manifestation of gut microbial dysbiosis induced by the chemotherapeutic 5-Fluorouracil (5-FU) in a validated in vitro model of the colon. BMC Microbiol. 2024;24(1):222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAaronson NK, Ahmedzai S, Bergman B, Bullinger M, Cull A, Duez NJ, et al. The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Cancer Inst. 1993;85(5):365\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanssen MF, Bonsel GJ, Luo N, Is. EQ-5D-5L Better Than EQ-5D-3L? A Head-to-Head Comparison of Descriptive Systems and Value Sets from Seven Countries. PharmacoEconomics. 2018;36(6):675\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrijsburg L, de Vries JH, Boshuizen HC, Hulshof PJ, Hollman PC, van 't Veer P, et al. Comparison of duplicate portion and 24 h recall as reference methods for validating a FFQ using urinary markers as the estimate of true intake. Br J Nutr. 2015;114(8):1304\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlagden SP, Charman SC, Sharples LD, Magee LR, Gilligan D. Performance status score: do patients and their oncologists agree? Br J Cancer. 2003;89(6):1022\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJukic A, Bakiri L, Wagner EF, Tilg H, Adolph TE. Calprotectin: from biomarker to biological function. Gut. 2021;70(10):1978\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRollenske T, Burkhalter S, Muerner L, von Gunten S, Lukasiewicz J, Wardemann H, et al. Parallelism of intestinal secretory IgA shapes functional microbial fitness. Nature. 2021;598(7882):657\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliphant K, Allen-Vercoe E. Macronutrient metabolism by the human gut microbiome: major fermentation by-products and their impact on host health. Microbiome. 2019;7(1):91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalstad TR, Bye A, Jenssen CR, Solheim TS, Thoresen L, Sand K. Patient interpretation of the Patient-Generated Subjective Global Assessment (PG-SGA) Short Form. Patient Prefer Adherence. 2019;13:1391\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCommon Terminology Criteria for Adverse Events (CTCAE) U.S.: National Institutes of Health _ National Cancer Institute. 2017 [Version v5.0:[Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm\u003c/span\u003e\u003cspan address=\"https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePallialine. Karnosfky Performance Score 2016 [Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.pallialine.nl/uploaded/docs/Depressie/Bijlage%20Karnofsky.pdf\u003c/span\u003e\u003cspan address=\"http://www.pallialine.nl/uploaded/docs/Depressie/Bijlage%20Karnofsky.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis SJ, Heaton KW. Stool form scale as a useful guide to intestinal transit time. Scand J Gastroenterol. 1997;32(9):920\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChow S-C, Wang H, Shao J. Sample Size Calculations in Clinical Research. 2nd ed2007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhavichitr N, Wang S, Chomto S, Tantibhaedhyangkul R, Kakourou A, Intarakhao S, et al. Impact of synbiotics on gut microbiota during early life: a randomized, double-blind study. Sci Rep. 2021;11(1):3534.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuet F, Abrahamse-Berkeveld M, Tims S, Simeoni U, Beley G, Savagner C, et al. Partly Fermented Infant Formulae With Specific Oligosaccharides Support Adequate Infant Growth and Are Well-Tolerated. J Pediatr Gastroenterol Nutr. 2016;63(4):e43\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc: Ser B (Methodol). 1995;57(1):289\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaş\u0026ccedil;ı E, Oyan B, S\u0026ouml;nmez \u0026Ouml;, Mutlu AU, Atcı MM, Sakin A, et al. Comparing the efficacy of regorafenib and 5-fluorouracil-based rechallenge chemotherapy in the third-line treatment of metastatic colorectal cancer. BMC Cancer. 2024;24(1):16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Liao X, Li Y, Cao H, Zhang F, Fei B, et al. Effects of prebiotic supplement on gut microbiota, drug bioavailability, and adverse effects in patients with colorectal cancer at different primary tumor locations receiving chemotherapy: study protocol for a randomized clinical trial. Trials. 2023;24(1):268.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUS National Library of Medicine ClinicalTrials. gov 2023 [Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/study/NCT05516641\u003c/span\u003e\u003cspan address=\"https://clinicaltrials.gov/study/NCT05516641\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiernikova S, Sevcikova A, Drgona L, Mego M. Modulating the gut microbiota by probiotics, prebiotics, postbiotics, and fecal microbiota transplantation: An emerging trend in cancer patient care. Biochim Biophys Acta Rev Cancer. 2023;1878(6):188990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams BA, Mikkelsen D, Flanagan BM, Gidley MJ. Dietary fibre: moving beyond the soluble/insoluble classification for monogastric nutrition, with an emphasis on humans and pigs. J Anim Sci Biotechnol. 2019;10:45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCantu-Jungles TM, Hamaker BR. New View on Dietary Fiber Selection for Predictable Shifts in Gut Microbiota. mBio. 2020;11(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAarnoutse R, Ziemons J, de Vos-Geelen J, Valkenburg-van Iersel L, Wildeboer ACL, Vievermans A, et al. The Role of Intestinal Microbiota in Metastatic Colorectal Cancer Patients Treated With Capecitabine. Clin Colorectal Cancer. 2022;21(2):e87\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsui D. Strategies to Measure and Improve Patient Adherence in Clinical Trials. Pharm Med. 2012;23(5\u0026ndash;6):289\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Colorectal neoplasms, Research design, Prebiotics, Microbiota","lastPublishedDoi":"10.21203/rs.3.rs-8436668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8436668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStandard systemic treatment for advanced colorectal cancer (CRC) often involves fluoropyrimidines, such as 5-fluorouracil (5-FU). Previous observational clinical studies, as well as pre-clinical research, demonstrated that chemotherapy affects the gut bacteria and its metabolites, potentially leading to microbial dysbiosis. Microbial dysbiosis might negatively influence anti-cancer efficacy and toxicity of the treatment. Therefore, targeted microbiota modulation using prebiotic fibers could potentially optimize 5-FU-based chemotherapy.\u003c/p\u003e\n\u003cp\u003eThis explorative intervention study aims to investigate the effects of daily administration of a prebiotic fiber mixture in a nutritionally complete oral nutritional supplement (ONS) on the intestinal microbiota composition in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy. Moreover, the effect of the prebiotic fiber ONS intervention on fecal, blood, clinical, and tolerance parameters will be evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e62 CRC patients scheduled for 5-FU-based therapy (FOLFOX, CAPOX, or capecitabine monotherapy) with or without bevacizumab, panitumumab or cetuximab will be prospectively enrolled in a double-blinded randomized controlled study. After informed consent, patients will be randomized to receive either the prebiotic fiber mixture ONS or control ONS, starting at least three days prior to the start of the 5-FU-based treatment. They will continue to take it twice daily for the duration of either four cycles of FOLFOX (in total 8 weeks) or three cycles of CAPOX or capecitabine monotherapy (in total 9 weeks). At multiple timepoints throughout the intervention, patients will collect fecal samples and complete questionnaires on food habits, patient characteristics, chemotherapy side effects, quality of life, current nutritional status, and physical performance. Microbiota composition, SCFA levels, and other parameters will be measured in the fecal samples. In addition, blood samples will be collected and analyzed for various parameters. Body composition and tumor response will be evaluated based on imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn conclusion, this randomized exploratory clinical intervention study outlines a novel approach to enhance the efficacy and reduce the toxicity of 5-FU-based chemotherapy in patients with advanced CRC through gut microbiota modulation using prebiotics. The knowledge obtained holds the potential to significantly impact clinical practices, improve patient outcomes, and advance microbiome-targeting interventions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is registered in the Dutch OMON database under NL86537.068.24\u003c/p\u003e","manuscriptTitle":"PREBICC - Prebiotic intervention in patients with advanced colorectal cancer treated with 5-FU-based chemotherapy: protocol of a double-blind randomized controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 16:09:35","doi":"10.21203/rs.3.rs-8436668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-06T14:39:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294448685432716190300510165865547442812","date":"2026-03-25T10:52:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T17:46:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221841920528514528308009434975255846040","date":"2026-03-18T15:16:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T15:11:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T23:56:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-23T23:55:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-12-23T18:51:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"37e5deca-d35c-4d2b-bfe3-c697535afa21","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-19T16:09:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 16:09:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8436668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8436668","identity":"rs-8436668","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}