Faecal microbiota transplantation for the treatment of diarrhoea induced by tyrosine-kinase inhibitors in patients with metastatic renal cell carcinoma

Diarrhoea is one of the most burdensome and common adverse events of chemotherapeutics, and has no standardised therapy to date. Increasing evidence suggests that the gut microbiome can influence the development of chemotherapy-induced diarrhoea. Here we report findings from a randomised clinical trial of faecal microbiota transplantation (FMT) to treat diarrhoea induced by tyrosine kinase inhibitors (TKI) in patients with metastatic renal cell carcinoma (ClinicalTrials.gov number: NCT04040712). The primary outcome is the resolution of diarrhoea four weeks after the end of treatments. Twenty patients are randomised to receive FMT from healthy donors or placebo FMT (vehicle only). Donor FMT is more effective than placebo FMT in treating TKI-induced diarrhoea, and a successful engraftment is observed in subjects receiving donor faeces. No serious adverse events are observed in both treatment arms. The trial meets pre-specified endpoints. Our findings suggest that the therapeutic manipulation of gut microbiota may become a promising treatment option to manage TKI-dependent diarrhoea.

Specific objectives or hypotheses 4

Trial design 3a
Description of trial design (such as parallel, factorial) including allocation ratio 10 3b Important changes to methods after trial commencement (such as eligibility criteria), with reasons N.A. Participants 4a Eligibility criteria for participants 10-11 4b Settings and locations where the data were collected 10 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered [11][12] Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 14 6b Any changes to trial outcomes after the trial commenced, with reasons N.A. Sample size 7a How sample size was determined 14-15 7b When applicable, explanation of any interim analyses and stopping guidelines N.A. Randomisation: Sequence generation 8a Method used to generate the random allocation sequence 14 8b Type of randomisation; details of any restriction (such as blocking and block size) 14 Allocation concealment mechanism 9 Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned

INTRODUCTION AND STUDY RATIONALE
Despite the improvement in diagnosis and management, renal cell carcinoma (RCC) remains one of the most burdensome urological cancers, being the sixth most common malignancy in men and the 10th in women, accounting, respectively, for 5% and 3% of all cancers (1). Moreover, the incidence of RCC is increasing, especially in Western countries (2), accounting for nearly 60000 new cases per year in the United States (3). A considerable proportion of patients present with metastatic disease at diagnosis (4,5), and there are more than 140000 RCC-dependent deaths per year worldwide according to the World Health Organization (6).
Sunitinib and pazopanib are oral multi-targeted receptor tyrosine kinase inhibitors (TKIs) that have dramatically improved the survival of patients with metastatic RCC (7), and are commonly used as first-line option for this condition (8).
However, long-term use of these drugs is prevented by the development of toxicity. Diarrhoea is one of the most common side effects of TKIs, occurring in nearly 50% of patients (9)(10)(11). It decreases the quality of life of these patients, and often requires dose reduction and drug discontinuation (12), potentially decreasing the efficacy of TKIs.
To date there are no standardised strategies for TKIs-related diarrhoea, and current recommendations are supported by few evidence or real-life experience. Recommended treatment options include anti-motility agents, which are not targeted to act on the pathogenic pathways of diarrhoea (13).
Increasing evidence suggests that gut microbiota could influence the development of TKIs-induced diarrhoea. Overall, chemotherapy is known to drive, through the development of mucositis, deep compositional and functional alterations of gut microbiota (14). Mucositis occurs commonly after treatment with TKIs (15), and a specific dysbiotic profile has been found in patients with TKIsinduced diarrhoea (16).
In theory, the therapeutic modulation of gut microbiota could be an approach to alleviate TKIinduced diarrhoea. Although probiotics have been suggested as a possible treatment option for this condition, few evidence supports this indication (17,18).

Faecal microbiota transplantation (FMT) is the infusion of faecal microbiota from a healthy donor
in the gut of a recipient with the aim of curing a specific disease. It has been increasingly recognised as a highly effective treatment against recurrent C. difficile infection (19,20).
FMT has been also examined as a potential approach for other disorders associated with a disruption of gut microbiota, including ulcerative colitis (21) or metabolic syndrome (22).
To date, the effects of FMT on chemotherapy-related diarrhoea are unknown. The aim of our study is to investigate the efficacy of faecal microbiota transplantation (FMT), compared with placebo FMT, in treating TKI-induced diarrhoea in patients with metastatic RCC.
• To assess the efficacy of donor FMT in treating TKI-induced diarrhoea • To investigate changes in gut microbiome after treatment in patients treated with donor FMT 3. METHODS

Study design
Single-centre placebo-controlled, double blind randomised clinical trial of donor FMT vs placebo FMT in patients with TKI-induced diarrhoea

Study population
Patients will be recruited among those referred to the oncology outpatient clinic of the Fondazione Policlinico Universitario "A. Gemelli". Patients with all inclusion criteria and none of the exclusion criteria will be considered for this study. • Blood exams, including: C reactive protein, erythrocyte sedimentation rate, transglutaminase antibodies, total IgA and IgE

Inclusion criteria
• Ileocolonoscopy All subjects who will meet eligibility criteria and will test negative for these exams will be finally enrolled in the study.

Baseline assessments
Before randomisation, demographic data will be collected by the oncology staff, and patients will be evaluated for the severity of diarrhoea according to the National Cancer Institute Common Toxicity Criteria; NCI CTC version 4.0 (grade 0 = none; grade 1 = increase of < 4 stools/day over pre-treatment; grade 2 = increase of 4-6 stools/day, or nocturnal stools; grade 3 = increase of ≥ 7 stools/day or incontinence or need for parenteral support for dehydration; grade 4 = physiologic consequences requiring intensive care, or hemodynamic collapse) (24).
Additionally, patients will be requested to give stool samples to be collected in a sterile, sealed container and stored at -80°C for metagenomic assessment of gut microbiome by the microbiology staff.

Treatments
After baseline assessments, patients will be randomly assigned to one of the following treatment arms: -Donor FMT (D-FMT) -Placebo FMT (P-FMT) Patients in both groups will undergo a single FMT procedure by colonoscopy.
Each patient in the donor FMT group will receive faeces from one single donor.
Placebo FMT will be made of 250 mL water.
Loperamide will be allowed as anti-diarrhoeal medication if diarrhoea will not respond to experimental treatments.

Selection of stool donors
The selection of stool donors will be performed by the gastroenterology staff following protocols previously recommended by international guidelines (20) The assignment of faecal infusates from healthy donors to patients will be done randomly, without any specific recipient-donor match, as this is not recommended by international guidelines (20).
Two healthy subjects will be selected, among donors from the donor bureau of the gastroenterology unit, as stool donors. At the time of their first donation, a sample of faeces will be collected and stored at -80°C for microbiome analysis.

Manufacturing of faecal infusates
All faecal infusates will be manufactured in the microbiology unit of our hospital. Only fresh faeces will be used. For each aliquot, 50 grams of faeces will be diluted in 250 mL of sterile saline. The deriving solution will be blended, and the supernatant strained and poured into a sterile container.

FMT procedures
All procedures will be performed by colonoscopy, under sedation. Patients in both groups wil undergo bowel cleansing with 4 litres of macrogol (SELG ESSE) the day before the procedure. All procedures will be performed by 2 expert endoscopists (G. C., L. R. L.), using pediatric colonoscopes and carbon dioxide insufflation. Both faecal infusates and placebo infusates will be delivered through the operative channel of the scope after reaching the more proximal point of the large bowel, using 50 mL syringes filled with the infusate during colonoscopy. The faecal infusate will be delivered within 6 hours after donor supply. After the procedures, patients will be monitored in the recovery room of the endoscopy centre for nearly 3 hours.

Follow-up
Follow-up visits will be performed by physicians from the oncology unit. All patients will be followed up for 2 months after the end of treatments. Follow-up visits will be scheduled at week 1, week 2, week 4, and week 8, after the end of treatments, respectively. At each visit the following assessments will be performed: 1) evaluation of the severity of diarrhoea, following the National Cancer Institute Common Toxicity Criteria (NCI CTC) version 4.0 (24); 2) record of concomitant medication, including loperamide; 3) collection of stool samples; 4) record of adverse events.
Unscheduled follow-up visits will be offered if requested by the patients. All adverse event occurred during follow-up will be recorded

Randomization and treatment allocation
Blocked randomisation of subjects will be performed by an external individual not involved in the study. An online random number generator software (https://www.sealedenvelope.com/simplerandomise r/v1/lists) will be used. To mask treatments to recipients, both infusates bottles and syringes will be covered with dark colored paper before the infusion, and the patients will be unable to see the endoscopic display during the procedure, which will be done under sedation. Moreover, the physicians who will visit patients at follow-up will be not aware of the treatment being administered.

Gut microbiota analysis
Gut microbiota analysis will be performed via shotgun metagenomics. Whole DNA will be extracted with the DNA was extracted using the Danagene Microbiome Fecal DNA kit and sequenced on the Illumina NovaSeq platform at an average of at least 4.5Gb. Default quality control will be done following the recommended and validated bioBarkery workflow, and resulting samples analyzed with the latest releases of MetaPhlAn for taxonomic profiling and StrainPhlAn for strainlevel profiling.

Sample size
To calculate sample size, we assume a 20% resolution rate of diarrhoea in the placebo arm (21) and a 80% resolution rate of diarrhoea in the FMT arm at 4 weeks of follow-up. Using a two-tailed a value of 0.05 and a power of 80% (b = 0.20), the enrolment of 10 patients per group is required.

Statistical analysis
The statistical analysis will be performed both on an intention-to-treat and per-protocol basis.
Differences among groups will be assessed with a two-tailed Wilcoxon-rank sum test for continuous data and with Fisher's exact probability test (using two-tailed P-values) for categorical data. Differences in cure percentages will be determined with Fisher's exact test (with two-tailed P values). For microbiome analysis, statistical differences between group means will be calculated using a two-tailed Wilcoxon-Rank Sum Test, through the R statistical software package (R Core Team, Vienna, Austria).

Safety Reporting
No specific serious adverse events are expected. Adverse events are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to the surveillance protocol. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded and reported to the coordinating investigator.
A serious adverse event is any untoward medical occurrence or effect that at any dose: • results in death; • is life threatening (at the time of the event); • requires hospitalisation or prolongation of existing inpatients' hospitalisation; • results in persistent or significant disability or incapacity; • is a congenital anomaly or birth defect; • is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the treatment of a life threatening disease, major safety finding from a newly completed animal study, etc.
All SAEs will be reported by the coordinating investigator.
SAEs that result in death or are life threatening should be reported expedited. The expedited reporting will occur not later than 7 days after the responsible investigator has first knowledge of the adverse reaction. This is for a preliminary report with another 8 days for completion of the report.
All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.

Ethics
The study protocol must be approved by the ethics committee of the Fondazione Policlinico Universitario "A. Gemelli" IRCCS, and will be registered at ClinicalTrials.gov. The study will be conducted in accordance with the Consolidated Standards of Reporting Trials (CONSORT) Statement