Measuring Effects of High-fiber Dried Chicory Root on the Gut Microbiota of Patients With an Intermediate to High-risk Cutaneous Melanoma: an Explorative Study
Phase: NA
Conditions: Melanoma, Stage II
Sponsor: University Medical Center Groningen

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MELFIB STUDY 
 
Measuring effects of high-fiber dried chicory root on the gut microbiota of 
patients with an intermediate to high-risk cutaneous melanoma:  
an explorative study 
 
RESEARCH PROTOCOL 
Hoofdaanvrager: Prof. dr. G.A.P. Hospers 
Universitair Medisch Centrum Groningen 
 
February 2025 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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‘Measuring effects of high-fiber dried chicory root on the gut microbiota of patients with an 
intermediate to high-risk cutaneous melanoma: an explorative study.’ 
Protocol ID 
19789 
Short title 
Explorative study on addition of high-fiber dried chicory root 
in patients with an intermediate to high-risk cutaneous 
melanoma. 
Version 
4 
Date 
February 27, 2025 
Principal investigators (in Dutch: 
hoofdonderzoekers/uitvoerders) 
 
Prof. Dr. G.A.P. Hospers 
University of Groningen 
University Medical Center Groningen 
Department of Medical Oncology 
g.a.p.hospers@umcg.nl 
Phone: +31652724185 
 
Investigators (in Dutch: 
uitvoerders) 
 
Emily Oosterhout, PhD candidate 
University of Groningen 
University Medical Center Groningen 
Department of Medical Oncology 
e.oosterhout@umcg.nl 
Phone: +31652764949 
 
Prof. Dr. B.L. van Leeuwen 
University of Groningen 
University Medical Center Groningen 
Department of Surgical Oncology 
b.l.van.leeuwen@umcg.nl  
Phone: +31631623706 
 
Prof. Dr. R.K. Weersma 
University of Groningen 
University Medical Center Groningen 
Department of Gastroenterology and Hepatology 
r.k.weersma@umcg.nl 
Phone: +31655256256 
 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
University Medical Center Groningen (UMCG) 
Hanzeplein 1, 9713 GZ Groningen 
Phone: +31503616161 
Subsidizing parties 
Seerave foundation 
WholeFiber Holding BV (in kind) 
Independent expert 
Dr. T.J.N. Hiltermann 
University of Groningen 
University Medical Center Groningen 
Department of Pulmonology and Tuberculosis 
t.j.n.hiltermann@umcg.nl 
Phone: +31652724086 

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PROTOCOL SIGNATURE SHEET 
Name 
Signature 
Date 
Head of Department: 
Prof. Dr. J.A. Gietema 
University of Groningen 
University Medical Center Groningen 
Department of Medical Oncology 
 
 
Principal Investigator: 
Prof. Dr. G.A.P. Hospers 
University of Groningen 
University Medical Center Groningen 
Department of Medical Oncology 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE .......................................................................................... 8 
1.1. 
Gut microbiome and dietary fiber ............................................................................................... 8 
1.2. 
Short-chain fatty acids, inulin and the immune system ............................................................... 8 
1.3. 
Gut microbiome and cancer ....................................................................................................... 8 
1.4. 
Target population: stage II melanoma ........................................................................................ 9 
1.4.1 
The Dutch population does not meet fiber intake recommendations .......................................... 9 
1.4.2 
Product: dried chicory root ....................................................................................................... 10 
1.5 
Future perspectives: moving towards advanced melanoma treated with immune checkpoint 
inhibitors .................................................................................................................................. 10 
2. 
OBJECTIVES .......................................................................................................................... 11 
3. 
STUDY DESIGN ..................................................................................................................... 12 
4. 
STUDY POPULATION ............................................................................................................ 13 
4.1. 
Population (base) .................................................................................................................... 13 
4.2. 
Inclusion criteria ...................................................................................................................... 13 
4.3. 
Exclusion criteria ..................................................................................................................... 13 
4.4. 
Sample size calculation ........................................................................................................... 13 
5. 
TREATMENT OF SUBJECTS ................................................................................................. 14 
6. 
INVESTIGATIONAL PRODUCT .............................................................................................. 15 
6.1. 
Name and description of the investigational product ................................................................ 15 
6.2. 
Summary of findings from non-clinical studies ......................................................................... 15 
6.3. 
Summary of findings from clinical studies ................................................................................ 15 
6.4. 
Summary of known and potential risks and benefits ................................................................ 15 
6.5. 
Description and justification of route of administration and dosage .......................................... 16 
6.6. 
Dosages, dosage modifications and method of administration ................................................. 16 
6.7. 
Preparation and labelling of Investigational Medicinal Product ................................................. 16 
6.8. 
Drug accountability .................................................................................................................. 16 
7. 
NON-INVESTIGATIONAL PRODUCT ..................................................................................... 17 
8. 
METHODS .............................................................................................................................. 18 
8.1. 
Study parameters .................................................................................................................... 18 
8.1.1. Main study parameter .............................................................................................................. 18 
8.1.2. Secondary study parameters ................................................................................................... 18 
8.1.3. Other study parameters ........................................................................................................... 18 
8.2. 
Randomization, blinding and treatment allocation .................................................................... 18 
8.3. 
Study procedures .................................................................................................................... 18 
8.3.1. Screening ................................................................................................................................ 18 
8.3.2. Assessment of medical history and demographics ................................................................... 19 
8.3.3. Blood sample collection ........................................................................................................... 19 
8.3.4. Laboratory assessments ......................................................................................................... 19 

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8.3.5. Fecal sampling ........................................................................................................................ 20 
8.3.6. Analyses of fecal material ........................................................................................................ 20 
8.3.7. Questionnaires ........................................................................................................................ 20 
8.3.8. Storage of samples ................................................................................................................. 20 
8.4. 
Withdrawal of individual subjects ............................................................................................. 20 
8.5. 
Specific criteria for withdrawal ................................................................................................. 21 
8.6. 
Replacement of individual subjects after withdrawal ................................................................ 21 
8.7. 
Premature termination of the study .......................................................................................... 21 
9. 
SAFETY REPORTING ............................................................................................................ 22 
9.1. 
Temporary halt for reasons of subject safety ........................................................................... 22 
9.2. 
AEs, SAEs and SUSARs ......................................................................................................... 22 
9.2.1. Adverse events (AEs) .............................................................................................................. 22 
9.2.2. Serious adverse events (SAEs) ............................................................................................... 22 
9.3. 
Follow-up of adverse events .................................................................................................... 22 
10. 
STATISTICAL ANALYSIS ....................................................................................................... 23 
10.1. 
Primary study parameter(s) ..................................................................................................... 23 
10.2. 
Secondary study parameter(s)................................................................................................. 23 
10.3. 
Other study parameters ........................................................................................................... 23 
11. 
ETHICAL CONSIDERATIONS ................................................................................................ 24 
11.1. 
Regulation statement............................................................................................................... 24 
11.2. 
Recruitment and consent ......................................................................................................... 24 
11.3. 
Benefits and risks assessment ................................................................................................ 24 
11.4. 
Compensation for injury ........................................................................................................... 25 
11.5. 
Incentives ................................................................................................................................ 25 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ........................................ 26 
12.1. 
Handling and storage of data and documents .......................................................................... 26 
12.2. 
Monitoring and quality assurance ............................................................................................ 26 
12.3. 
Amendments ........................................................................................................................... 27 
12.4. 
Annual progress report ............................................................................................................ 27 
12.5. 
Temporary halt and (prematurely) end of study report ............................................................. 27 
12.6. 
Public disclosure and publication policy ................................................................................... 27 
13. 
STRUCTURED RISK ANALYSIS ............................................................................................ 28 
13.1. 
Potential issues of concern ...................................................................................................... 28 
13.2. 
Synthesis ................................................................................................................................ 28 
14. 
REFERENCES ........................................................................................................................ 29 
15. 
APENDIX ................................................................................................................................ 31 
15.1. 
Flow chart of study procedures ................................................................................................ 31 
15.2. 
Fecal sampling ........................................................................................................................ 32 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
AE 
Adverse Event 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
ELISA 
Enzyme-Linked Immuno Sorbent Assay 
FDR 
False Discovery Rate 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
METc  
Medical research ethics committee (MREc); in Dutch: medisch-ethische 
toetsingscommissie (METc) 
PBMC 
Peripheral Blood Mononuclear Cells 
PIF 
Patient Information Form 
(S)AE 
(Serious) Adverse Event  
SCFA 
Short Chain Fatty Acid 
SD 
Standard Deviation 
SEM 
Standard Error of the Mean 
SPC 
Summary of Product Characteristics; in Dutch: officiële productinformatie 
IB1-tekst 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 

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SUMMARY 
Rationale: Dietary fibers are digested in the gut by specific bacteria, which can produce short-chain 
fatty acids (SCFAs). These SCFAs together with dietary fiber on its own are hypothesized to have a 
beneficial effect on the immune cell composition, fecal calprotectin levels, stool pattern and general 
well-being. In intermediate to high-risk cutaneous melanoma (minimal stage II A) very often tumor 
infiltrating lymphocytes are found, which sometimes leads to spontaneous remission of these tumors. 
What is unclear, and in our view should be the first small step and the objective of this study, is to 
determine whether changes in the gut microbiome in this group of patients (with skin melanoma) is 
possible at all, if these changes in the gut microbiome lead to an increased production of SCFA and 
whether the changes in gut microbiome; immune cell composition and SCFA can indeed be 
measured. 
Objective: Exploring the effect of supplementation with daily addition of prebiotic dried chicory root 
(WholeFiberTM) for 6 weeks on fecal SCFA levels, gut microbial composition and immune cell 
composition in minimal stage II A melanoma patients following surgical treatment.  
Study design: Supplementing with WholeFiberTM for 6 weeks two times a day (10 g per portion). 
During 3 visits questionnaires, measurements and (blood and fecal) samples will be taken.  
Study population: The research population will be recruited at the University Medical Center 
Groningen (UMCG) and consists of 20 patients aged ≥18 years, who have undergone surgical 
treatment of a minimal stage II A melanoma, with no concurrent adjuvant treatment or autoimmune 
disease.  
Intervention: Subjects will receive 2 sachets of 10 g WholeFiberTM per day for 6 weeks (equals 17 
g fiber). In the first week of the intervention, the subjects will take 1 sachet of 10 g WholeFiberTM 
(equals 8 g fiber).  
Main study parameters: The main study parameter is determining the effect of WholeFiberTM for 6 
weeks on fecal SCFA levels in this patient group. Secondary (exploratory) parameters are gut 
microbial composition changes, immune cell composition, stool patterns, fecal calprotectin levels, 
and side effects after intervention. 
Nature and extent of the burden and risks associated with participation, benefit, and group 
relatedness: WholeFiberTM is a dried vegetable (chicory root) that for 85% consists of prebiotic 
fibers, safe for consumption and is commercially available. Increasing dietary fiber intake is beneficial 
for overall health and may give some abdominal complaints at the start. Patients will visit the study 
site three times for blood tests and fecal samples will be collected at home. The collected fecal 
samples will be brought to the study visit by the participant to ensure the lowest impact for the 
subjects. 
 
 

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1. INTRODUCTION AND RATIONALE 
1.1. Gut microbiome and dietary fiber  
Chronic diseases such as diabetes, inflammatory bowel disease and cancer have dramatically 
increased over the past century, many of which are associated with changes in the gut microbiome1. 
This suggests that certain lifestyle changes may disrupt gut homeostasis and cause microbial 
dysbiosis; a condition typically characterized by the growth of pathogens at the expense of 
commensal bacteria when compared to a symbiotic microbiome2. One of the main factors affecting 
the composition of the gut microbiome is diet. Research has shown that the occurrence of beneficial 
microbes is often linked to food items rich in poly-unsaturated fatty acids, dietary fiber, and 
polyphenols6. These compounds are found in great amounts in the Mediterranean diet3. 
Despite these well described correlations, the associated dietary patterns are not yet tested in a 
clinical setting. Prospective clinical studies using a prescribed diet are hard to perform and to 
analyze4. Therefore, use of add-on dietary fiber could serve as an alternative approach to study 
these so-called beneficial dietary patterns. 
 
1.2. Short-chain fatty acids, inulin and the immune system 
Dietary fibers are digested in the intestine by specific microbes, which produce short-chain fatty acids 
(SCFAs) such as acetate, propionate and butyrate as a byproduct. SCFAs have beneficial effects 
on gut health by acting as anti-inflammatory compounds and increasing gut-barrier function5. 
They also help modulating immune responses by interacting with various immune cells6. SCFAs 
such as acetate, propionate and butyrate have been identified to promote generation of colonic 
regulatory T cells9. Regulatory T cells produce several inhibitory cytokines including TGF-ß, IL-35 
and IL-10 (Figure 1)7,8. SCFAs can also enter systemic circulation, thus affecting immune cells 
beyond the gut9. 
The dietary fiber inulin also plays an important role in regulating immune cell differentiation. The 
prebiotic fiber present in chicory root has been shown to increase the production of TNF- and IFN-
 by CD4+ T cells (Figure 1)10. These immune cells are also known as T helper (TH) cells, which are 
important in almost all adaptive immune responses. Inulin also directly affects CD8+ T cells and  
T cells, which are innate immune cells who can directly kill infected, cancerous, or non-self-cells11–
13.  
 
1.3. Gut microbiome and cancer 
A recent review shows indications of gut OncoMicrobiome signatures, suggesting that patients with 
melanoma may have a different microbiome14. Additionally, there are ongoing studies on altering the 
microbiome in melanoma patients undergoing immunotherapy with the goal to make immunotherapy 
more effective. The methods that are studied now are fecal transplantation and nutritional 
modulations15–17. We know that the intake of dietary fiber can affect gut microbial composition18. 
Therefore, our proposal is to conduct this exploratory study using dietary fiber in patients with “early 
melanoma” to investigate the axis between fiber intake, gut microbiome and immune cell 
composition. In these patients, there are fewer confounders (no immunotherapy, colitis, steroids, 
etc.), which may contribute to better insight into the microbiome in these patients, the effect of 
increasing fiber on the gut microbiome and metabolites, and the immune cell composition. Moreover, 
we are gaining more information about the “healthy microbiome” through data from the Lifelines 
database in several ongoing studies, secondly these data could serve as a control. For example, we 
are investigating whether there is a relationship between gut microbiome and the development of 
melanoma (and other tumors) using the link between the Lifelines database and the Pathological-
Anatomical National Automated Archive (PALGA) database. 

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Figure 1: Proposed mechanism of action of WholeFiberTM on cellular level in the gut. 
 
1.4. Target population: stage II melanoma 
In intermediate to high-risk cutaneous melanoma (minimal stage II A) lymphocytes are often found, 
which may lead to spontaneous remission of these tumors19. In line with these findings, ICI treatment 
has been shown to be effective in this patient group as the 5–15-year survival rate of patients with 
this tumor type in stage IV strongly improved after ICI treatment20. This type of cancer treatment 
helps immune cells recognize tumor cells, enabling targeted elimination. Therefore, it can be 
beneficial to explore the effects of an inulin rich fiber in this population, as this has been shown to 
affect immune cell composition.  
 
1.4.1 The Dutch population does not meet fiber intake recommendations  
A recent study by Reynolds et al investigated the relationship between carbohydrate quality and 
global human health and found that implementation of recommendations to increase dietary fiber 
intake benefits human health21. The current guideline for fiber consumption in the Netherlands is a 
minimal intake of 14 grams of fiber per 1000 kcal consumed in adults aged 21-50, which translates 
to about >25 grams of fiber per day for women and >38 grams of fiber per day for men22. Interestingly, 
the general Dutch population does not meet these recommendations, as the mean fiber intake in the 
this population was measured at 20.3 g/day by the National Food Consumption Survey of 2019-
202122.  
Implementation of high fiber food sources in meals throughout the day would pose a solution for the 
stated ‘fiber intake gap’. However, it remains difficult to increase fiber intake, even with personalized 
advice, and easy-add-ons in the diet such as a supplement are suggested to have a better 
adherence4. Therefore, there is a need for an easy solution to increase fiber intake it in an effective 
way to meet the recommended dietary intake of fiber21.  
 

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1.4.2 Product: dried chicory root 
In this study, we propose to use WholeFiberTM as an easy add-on to the daily diet. WholeFiberTM is 
100% dried root vegetable, chicory root, in which 85% of its dry weight consists of fiber. By using 20 
g of the product daily the earlier mentioned ‘fiber intake gap’ can already be tackled. The fiber 
composition of chicory root contains multiple intrinsic prebiotic fibers, mainly inulin, and some pectin, 
hemi-cellulose, and cellulose. Furthermore, this dried vegetable is rich in polyphenols and 
potassium.  
The dried vegetable WholeFiberTM has been used in a recent 5-week randomized controlled trial with 
Dutch adults at high risk of developing type 2 diabetes. In the study, 15-30 g/day of WholeFiberTM 
has been shown to significantly modulate the gut microbiota composition18. More specifically, they 
measured a great impact of WholeFiberTM on butyrate-producing bacteria such as Bifidobacterium 
and Anaerostipes (3-4-fold increase), as well as increased SCFA levels in feces (+26%)18.  
In a different trial where 15-30 g of WholeFiberTM was consumed daily for 12 weeks, an improvement 
of 
insulin 
sensitivity, 
plasma 
triglycerides, 
fat 
cells 
and 
liver 
fat 
(https://classic.clinicaltrials.gov/ct2/show/NCT04714944) 
in 
40 
overweight 
insulin 
resistant 
participants was measured. Furthermore, similar impact of WholeFiberTM on the gut microbiota, e.g. 
increase in Bifidobacterium and Anaerostipes, and SCFA levels was seen (data not published yet). 
Both studies show that WholeFiberTM modulates the gut microbial composition and promotes SCFA 
production, which can be explained due to the intrinsic nature of the fibers in WholeFiberTM. These 
fibers are still in the original plant cell structure, as the chicory root is hardly processed17. Therefore, 
WholeFiberTM partly reaches the distal colon, where fibers have the largest impact on the gut 
microbiota, SCFA levels and immune cell composition23.  
We are planning to conduct this study in patients with a cutaneous melanoma (T3a, T3b, T4a, T4b, 
N0 or N+, M0, Stage IV with no evidence of M+ disease), that have undergone curative surgical 
treatment and will not undergo (neo)adjuvant treatment. The product that we are going to use in this 
trial, WholeFiberTM, is hypothesized to influence the microbial composition in a way that specific 
microbes, which are reported to produce SCFA, are favored on short term16,19. The goal of the study 
is to test the effect of the inulin rich dietary fiber without any conditions that are hypothesized to 
influence its effects24,25. The proposed amount of 20 g has been used in previous studies using 
WholeFiberTM, where no adverse effects were reported26.  
 
1.5 Future perspectives: moving towards advanced melanoma treated with immune checkpoint 
inhibitors 
The discovery of the link between the gut microbiome, diet and response to immune checkpoint 
inhibitors (ICI) in stage IV melanoma patients have shown that the gut microbiome is not only a 
biomarker for treatment response but can also be a modifiable treatment target27,28. Interestingly, the 
immune system plays an important role in patients with melanoma tumors.  
What is unclear, and in our view should be the first small step and the objective of this study, is to 
determine whether changes in the gut microbiome in this group of patients (with skin melanoma) is 
possible at all, if these changes in the gut microbiome lead to an increased production of SCFA and 
whether the changes in gut microbiome; immune cell composition and SCFA can indeed be 
measured. 
 
 
 
 
 
 
 
 

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2. OBJECTIVES 
The primary objective of this study is to investigate the effect of dried chicory root (WholeFiberTM) for 
6 weeks on fecal SCFA levels in intermediate to high-risk melanoma patients after surgical treatment 
with no concurrent adjuvant treatment. 
 
Secondary objectives 
• 
Exploration of the gut microbial composition of surgically treated intermediate to high-risk 
cutaneous melanoma patients before intake and potential compositional changes after using 
WholeFiberTM for 6 weeks.  
• 
Exploration of the effect of WholeFiberTM use for 6 weeks on immune cell composition, stool 
patterns, fecal calprotectin levels and side effects in intermediate to high-risk cutaneous 
melanoma patients after surgical treatment, with no concurrent adjuvant treatment. 
 
 

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3. STUDY DESIGN 
This study will be an explorative study at the University Medical Center Groningen (UMCG). All 
measurements, in detail described in chapter ‘8.3 – Study procedures’, will be performed in all 
patients. The effect of oral intake of dried chicory root (WholeFiberTM) in intermediate to high-risk 
cutaneous melanoma after surgical treatment, with no concurrent adjuvant treatment, will be tested. 
Inclusion of patients in the study can take place within 16 weeks after surgery, the baseline visit (T0) 
can take place later than 16 weeks after surgery. This is in line with the exclusion criterium that 
patients can’t use antibiotics in the 3 months prior to inclusion. WholeFiberTM will be used two times 
a day (10 g per portion) by the patients for 6 weeks. In the first week of the intervention WholeFiberTM 
will be used one time a day (10 g per portion) to let the study participants adjust to the product. The 
study consists of 4 visits, including the informed consent visit. The estimated visit time is 60 minutes, 
apart from the inclusion visit which will take approximately 30 minutes.  
 
Three timepoints are included in this study (Figure 2): 
• 
Baseline (T0), where patients will collect a fecal sample at home, a blood sample will be drawn, 
and patients will fill in questionnaires. 
• 
Mid-Trial (T1), collection of a fecal sample at home, a blood sample will be drawn, and patients 
will fill in questionnaires. 
• 
End of Trial (T2), collection of a fecal sample at home, a blood sample will be drawn, and patients 
will fill in questionnaires. 
 
Figure 2: Trial design with 3 timepoints and specification about the measurements + questionnaires per time point. 
 
 
 
 

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4. STUDY POPULATION 
4.1. Population (base)  
The research population consists of intermediate to high-risk cutaneous melanoma patients who 
received surgical treatment at the University Medical Center Groningen (UMCG), that will not receive 
adjuvant treatment during the study period. Patients will be notified about the study at the UMCG by 
their treating physician and informed about the study by the coordinating investigator.  
4.2. Inclusion criteria 
To be eligible to participate in this study, a subject must meet all the following criteria: 
• 
Age ≥18 years; 
• 
The participant understands the study and can provide written informed consent; 
• 
The participant received surgical treatment of an intermediate to high-risk cutaneous 
melanoma (T2a, T2b, T3a, T3b, T4a, T4b, N0 or N+, M0, Stage IV with no evidence of M+ 
disease); 
• 
Being able to read and speak Dutch; 
• 
Willing to come to the UMCG for practical reasons (visiting the study site); 
• 
Willing to continue their regular lifestyle patterns during the study. 
4.3. Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation in this 
study: 
• 
Receiving concurrent adjuvant treatment, adjuvant treatment after the study period is 
allowed.  
• 
Having a medical history that may impact study outcomes, such as a diagnosis of diabetes 
mellitus type 2, heart disease, renal disease, autoimmune disease; 
• 
Any clinically significant or unstable medical disorder involving the gut, including celiac 
disease, inflammatory bowel disease, short-bowel syndrome or acute/chronic pancreatitis; 
• 
Having an ileostomy or colostomy, as this greatly impacts bowel function and gut microbial 
composition; 
• 
Use of antibiotics in the 3 months prior participation in the study; 
• 
Use of prednisolone or other immunosuppressive medication; 
• 
Use of tube feeding or sib-feeding; 
• 
Being pregnant or lactating; 
• 
Participation in another interventional study at the same time; 
• 
Unable or unwilling to comply to study rules. 
4.4. Sample size calculation 
A sample size calculation was not performed for this study since it is an explorative study. We do 
not aim to reach statistical significance, paired differences with a 95% confidence interval will suffice 
to reach the aim of the study. The main aim is to explore the effects of the intervention on our 
parameters and answer tolerability questions. Since this is an explorative study, inclusion of 20 
participants should be sufficient10.  
 
 
 
 

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5. TREATMENT OF SUBJECTS 
We intent to use the food product WholeFiberTM (WholeFiber Holding BV, Espel, Netherlands) in the 
proposed trial. This product is commercially available on the Dutch market. The product consists of 
100% chicory root and contains all the macro and micro-nutrients found in this root. WholeFiberTM is 
a vegetable product with high levels of prebiotic intrinsic dietary fibers (~85%) mainly consisting of 
inulin, and some pectin, hemi-cellulose and cellulose that is derived from the root vegetable, chicory 
roots. The dose will be 20 g/day corresponding to 17 g/day of dietary fiber, except for the first week 
where 10 g/day will be used. Besides dietary fibers, it contains low levels of protein (5%), fat (<1%), 
mono- and disaccharides (3%), organic acids (2%) and minerals (2%). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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6. INVESTIGATIONAL PRODUCT  
6.1. Name and description of the investigational product 
WholeFiber is a vegetable product with high levels of prebiotic intrinsic dietary fibers (~85%) mainly 
consisting of inulin, and some pectin, hemi-cellulose and cellulose that is derived from chicory roots. 
For the production, the roots have been cut in small cubes and dried in specific conditions, resulting 
in granules with a size of ~4mm. Chicory roots are a major source of inulin and is therefore widely 
used for inulin extraction23. WholeFiber is produced in the Netherlands and commercially available. 
WholeFiber Holding BV is a certified food producer and is FSC22000 certified. 
6.2. Summary of findings from non-clinical studies 
Multiple mouse and rat studies have been performed, investigating the effect of dietary inulin on the 
ability to prevent colonization by pathogenic species24,25. Inulin-type fructans increase height of villi, 
depth of the crypts, number of goblet cells and thickness of the epithelial mucus layer in a study 
comparing germ-free rats and rats with a human fecal flora26. An important finding of this study was 
the observation that inulin-type fructans stimulated the number of bifidobacteria in the mucosa-
associated flora, as assessed by molecular techniques based on fluorescent in situ hybridization 
with 16s rRNA-targeted probes. One in vitro fermentation study has been published using 
WholeFiberTM that found an increase in butyrate production, a SCFA that is produced by the gut 
microbiota and is associated with positive health effects27. 
6.3. Summary of findings from clinical studies 
WholeFiber has been investigated in a five-week investigator-blinded randomized controlled trial 
including 55 subjects at risk for type 2 diabetes mellitus by Wageningen University16. Subjects first 
consumed 2 weeks of 15g/day of WholeFiber or 8 g of maltodextrin, followed by a three-week 
consumption of 30 g/day of WholeFiber or 16 g/day of maltodextrin. WholeFiber strongly 
modulated gut microbiota composition (7% variation, p = .001) and dramatically increased relative 
levels (3-4-fold) of Anaerostipes and Bifidobacterium spp., in a dose- dependent, reversible manner. 
Fecal SCFA levels such as butyrate also increased dramatically (+25.8%, p = .023). Furthermore, 
stool softness and frequency increases, and glycemic coefficient of variation decreased. In another 
trial by Maastricht University, 40 subjects at risk of type 2 diabetes consumed 15-30 g/day of 
WholeFiber for 12 weeks in a double-blind randomized controlled trial and was compared to 
maltodextrin. It was found that WholeFiberTM improved insulin sensitivity (measured by a 2-step 
hyperinsulinemic clamp), plasma triglycerides and liver fat content, which was not the case for 
placebo (in-house data, not published yet). Data-analysis of this trial is still ongoing as data collection 
was completed in March 2023 (https://classic.clinicaltrials.gov/ct2/show/NCT04714944). The 
WholeFiber intervention was well tolerated in both trials. Therefore, we propose that using 20 g of 
the product daily will be tolerated in our patient group.  
6.4. Summary of known and potential risks and benefits 
WholeFiberTM consists for 85% out of dietary fibers. Research participants who do not normally have 
a high-fiber diet may experience gastrointestinal symptoms (e.g. bloating, flatulence, diarrhea, 
constipation, or cramping) for a maximum of two weeks when they consume WholeFiberTM. These 
discomforts are minor and should be limited to the first few days after starting to use the product. 
These discomforts should not last longer than a few hours per day. In the previous clinical trials, the 
participants did not report any major AEs in any of the study visits. Potential benefits are related to 
reported benefits of increasing fiber intake (improving stool pattern, improving glycemic control, and 
modulating the gut microbiota).  
 

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6.5. Description and justification of route of administration and dosage 
The product is processed into food cubes and will be delivered in a commercially available package 
containing 350 g. The sachets containing a dose of 10 gram will be packaged by the research team 
member together with the participant during the first visit. This will be taken twice a day, adding up 
to a daily dose of 20 grams. In the first week participants will take a daily dose of 10 grams, to enable 
the participants to adjust to the product. Each sachet of WholeFiberTM consists of 10 grams 
WholeFiberTM. In the study of Puhlmann et al (2022) and the Maastricht Study, subjects consumed 
30 g/day of WholeFiberTM, which was well tolerated. Furthermore, the mean fiber intake in the Dutch 
population was measured at 20.3 g/day in the national food consumption survey of 2019-202114, 
using 20 g of WholeFiberTM daily will add enough fiber to the participant’s diet to meet the 
recommended daily fiber intake (35-40 g/day).  Participants can choose how they consume 
WholeFiberTM, this can be done as is or add/sprinkle it over their meal. Advice will be given how the 
product can be used for optimal compliance. 
6.6. Dosages, dosage modifications and method of administration 
Each sachet of WholeFiberTM consists of 10 grams of WholeFiberTM. Subjects will consume 2 sachets 
per day for 5 weeks, resulting in 20 g/day of WholeFiberTM. In the first week of the intervention period 
the participants consume 1 sachet per day, resulting in 10 g/day of WholeFiberTM. The participant 
will be advised to add the product to various dishes according to one’s taste. Subjects can use 
WholeFiberTM in the recipes if they wish, but it can also be consumed as is. Advice will be given on 
consumption of WholeFiberTM. 
6.7. Preparation and labelling of Investigational Medicinal Product 
Preparation and the labeling of WholeFiberTM (350 g) will be conducted at WholeFiber Holding BV 
(Espel, the Netherlands), the same packaging is used as the commercially available product from 
WholeFiber Holding BV. Packaging of the individual sachets (supplemented by WholeFiberTM) will 
be performed by the patient together with a member of the research team using a basetech scale 
(conrad article number: 1283451-83) to weigh out the portion size (10 g). Packaging of the products 
in sachets will be done according to the relevant GMP guidelines and takes place during the first 
visit. The sachets will be labelled with: Study name, content sachet (WholeFiberTM), research 
institute, expiration date and storing conditions. Subjects will receive an additional document stating 
the use of WholeFiberTM and the ingredients of the sachets. The outer box will contain contact details 
of the investigators.  
6.8. Drug accountability 
Patients receive the product at the first visit. The sachets (supplemented by WholeFiberTM) will be 
filled by the patient together with a member of the research team using a basetech scale (conrad 
article number: 1283451-83) to weigh out the portion size (10 g). At home, subjects are asked to 
store the sachets at room temperature for the duration of the intervention period. Each subject will 
receive 90 sachets (including 6 reserve sachets). At each visit, the patient is asked to bring the 
remaining sachets, and the researcher will count how many sachets are left. A calculation is made 
to estimate whether all sachets are used on each day.  
 
 
 
 
 
 
 
 
 
 
 
 

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7. NON-INVESTIGATIONAL PRODUCT 
 
Not applicable. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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8. METHODS 
8.1. Study parameters 
8.1.1. 
Main study parameter 
Effect of a high fiber prebiotic vegetable (WholeFiberTM) for 6 weeks on fecal SCFA levels in 
intermediate to high-risk cutaneous melanoma patients, with no concurrent adjuvant treatment. 
HPLC ion chromatography system (Metrohm AG, Herisau, Switzerland) will be used to 
determine the levels of SCFA. The concentrations of SCFA; butyrate, propionate, and acetate 
will be measured with a conductivity detector. SCFA will be measured at baseline, halfway 
intervention and after 6 weeks.  
8.1.2. 
Secondary study parameters  
Discovering the gut microbial composition changes measured in intermediate to high-risk 
cutaneous melanoma patients with no concurrent adjuvant treatment, after using WholeFiberTM 
for 6 weeks. This analysis will be based on whole genome sequencing of fecal samples 
performed at the UMCG according to standardized protocol.  
Exploring the effect of WholeFiberTM use for 6 weeks on immune functioning, stool patterns, 
fecal calprotectin levels and side effects in this patient group. Peripheral blood mononuclear 
cells (PBMCs) and bloodplasma will be isolated to determine immune cell composition before 
and after the intervention period. Fecal calprotectin will be determined using the respective 
ELISA kit. Calprotectin is released when neutrophils enter the gut wall and are activated during 
the inflammatory process28. Lastly, eating patterns, fiber intake before the interventional period, 
stool patterns and side effects will be identified using the Food Frequency Questionnaire29, 
Bristol Stool Scale30, Gastrointestinal Symptoms Rating Scale31 and Health Related Quality of 
Life32 questionnaires. 
8.1.3. 
Other study parameters  
Anthropometry measurements at the baseline visit such as height, weight, BMI, waist 
circumference. We will extract demographic and disease progression data from patients’ 
medical records. If data is missing in the medical records, additional information will be acquired 
by interviewing the patient during the clinical visit(s). 
8.2. Randomization, blinding and treatment allocation 
Not applicable, as it is an explorative study with one arm, where we are also interested in the 
tolerability of the product by this patient group. 
8.3. Study procedures 
8.3.1. 
Screening 
Surgically treated intermediate to high-risk cutaneous melanoma patients with no concurrent 
adjuvant treatment will be notified about this explorative study by their treating physician. When 
the patient is interested, the coordinating investigator can explain the study and provide the 
patient with the patient information form (PIF). The patient and the study coordinator can 
discuss about the next steps in study participation. Written informed consent for participation 
in the trial must be obtained before performing any study specific screening tests or evaluation. 
All screening evaluations must be completed and reviewed to confirm that patients meet all 
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8.3.2. 
Assessment of medical history and demographics 
Medical history includes clinically significant diseases, surgeries, cancer history (including prior 
cancer therapies and procedures), reproductive status, smoking history, and all medications 
(e.g. prescription drugs, over-the-counter drugs, herbal or homeopathic remedies, nutritional 
supplements) used by the patient within 3 months prior to the screening visit. Demographic 
data will include age, sex, BMI, and self-reported race/ethnicity. 
8.3.3. 
Blood sample collection 
Blood will be collected at the medical oncology department of the UMCG using a standardized 
protocol. The drawing of blood will be performed by trained employees and the time of sampling 
will be recorded on a blood drawing form. Blood will be drawn from the antecubital vein in the 
arm. During each visit, 10 ml of blood will be collected for PBMC isolation and plasma 
measurements. Specific collection tubes that are used are CPT tubes. 
Blood will then be transferred to the laboratory site and PBMC isolation will take place 
immediately. The average amount of cells that can be isolated is 1.30*106 PBMCs/ml. PBMCs 
and plasma will then be stored at -80ºC, at the same laboratory site as the isolation, until 
analysis. PBMCs will be tranfered to liquid nitrogen storage 24 hours after isolation of the 
PBMCs.  
8.3.4. 
Laboratory assessments 
Samples of venous blood drawn at each visit will be analyzed at the study site’s local laboratory. 
Local laboratory assessments will include PBMC isolation and storage of plasma. By using flow 
cytometric analysis the immune cell composition will be determined (Table 1).  
 
Table 1: Flowcytometry panel PBMC analysis 
Marker 
Fluorochrome Clone 
Cell Type 
Marker Type 
CD24 
BUV395 
ML5 
Monocytes 
Surface  
CD27 
BUV661 
M-T271 Memory T cells 
Surface  
CD45 
BUV737 
HI30 
Leukocytes 
Surface  
CD38 
BUV805 
HB7 
Activated T cells 
Surface  
CD3 
APC-H7 
SK7 
T cells, γδ T cells 
Surface  
CD45RA 
BV711 
HI100 
Naive T cells 
Surface  
CD4 
BV785 
OKT4 
Helper T cells 
Surface  
FoxP3 
AF488 
259D 
Regulatory T cells (Tregs) 
Intracellular  
CD8 
PerCP/Cy5.5 
SK1 
Cytotoxic T cells 
Surface  
CD14 
APC 
HCD14 
Monocytes 
Surface  
TNF 
PE 
MAb11 
Inflammatory cytokine 
Intracellular  
IFNγ 
PE/Cy7 
4S.B3 
Cytokine in T cells 
Intracellular  
CD25 
PE 
BC96 
Regulatory T cells (Tregs) 
Surface  
TCR γδ 
FITC 
B1.1 
γδ T cells 
Surface  
CD11c 
BV605 
3.9 
Dendritic cells, monocytes Surface  
CD16 
BV510 
3G8 
Non-classical monocytes 
Surface  
 
 
 
 
 
 

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8.3.5. 
Fecal sampling 
Three tubes of feces will be collected prior to the WholeFiberTM intervention, mid-trial, and at 
the end of the 6-week intervention period. After instructions, patients will collect their feces at 
home in a standardized manner. See section 15.2 for a detailed description of the stool 
sampling.  
The stool samples must be stored in a (home) freezer within 10 minutes after collection. At the 
study visit, the stool samples will be brought by the participant in a cooler bag. These 
procedures are based on protocols used in Lifelines. After processing, the samples will be 
stored at -80ºC at the Microbiome Hub (UMCG). 
8.3.6. 
Analyses of fecal material 
Collected tubes with feces samples will be stored (at -80°C) until a sufficient batch size is 
present for further analyses (calprotectin, microbiome, SCFAs). Analyses of the microbiome 
will be conducted within the UMCG following standardized procedures. The composition and 
function of the gut microbiome will be analyzed prior to, mid-trial, and after the WholeFiberTM 
intervention using whole genome sequencing. Temporal changes in the gut microbiome 
following the WholeFiberTM intervention can be traced up to species and even strain level by 
using novel algorithms33. Analyses and interpretation of the microbiome will be in the context 
of patient characteristics (age, sex, BMI), and use of medication, including vitamins, probiotics, 
and homeopathic compounds.  
HPLC ion chromatography system (Metrohm AG, Herisau, Switzerland) will be used for the 
short chain fatty acid analysis. The concentrations of SCFA (acetic acid, propionic acid, butyric 
acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, caproic acid) will be 
measured with a conductivity detector. The 6.1005.200 Metrosep Organic Acids column 
(Metrohm AG) with 5mm particle size and dimensions of 7.8 mm by 250 mm will be used for 
differentiating fatty acids. The mobile phase is 5mM HNO3 and acetonitrile v/v ratio of 98:2, 
respectively, with a flow rate of 0.6 ml per minute. 
Fecal calprotectin levels will be measured in the collected fecal samples (baseline, mid-trial, 
end of intervention) using Enzyme-Linked Immuno Sorbent Assay (ELISA). 
8.3.7. 
Questionnaires 
Subjects will complete the questionnaires prior to the study visit. After enrolment in the study, 
they will receive the questionnaires via post (physical or online), which need to be filled in prior 
to every study visit. This will take up to one hour. These questionnaires include: 
• 
Food Frequency Questionnaire29,34 (FFQ) 
• 
Health Related Quality of Life32 (HR-QoL) 
• 
Bristol Stool Chart30 
• 
Gastrointestinal Symptom Rating Scale31 (GSRS) 
8.3.8. 
Storage of samples 
All patient related material (blood and feces) will be l be anonymized by using a unique patient 
identification code (see also section 12.1) 
8.4. Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for urgent medical 
reasons. If subjects leave the study, their results (blood samples, fecal samples, and questionnaires) 
will be used for the analyses unless the patient specifically states that we may not use the collected 
data and material when signing the informed consent. 

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8.5. Specific criteria for withdrawal 
Reasons for withdrawal from the study may include but are not limited to the following: 
• 
Subject withdrawal of consent at any time; 
• 
Any medical condition that the investigator determines may jeopardize the subject’s safety if 
he or she continues in the study; 
• 
Investigator determines it is in the best interest of the subject; 
• 
Subject non-compliance. 
8.6. Replacement of individual subjects after withdrawal 
If subjects withdraw before the intervention has started, they will be replaced. 
8.7. Premature termination of the study 
The study is a short intervention study and we do not expect a premature termination. Since 
WholeFiberTM is suitable for human consumption, safe, and commercially available, we do not expect 
any negative events. Consumption of the product may result in uncomfortable though harmless 
effects such as increased flatulence or bloating. Research has shown that these adverse events are 
highly variable among people. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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9. SAFETY REPORTING 
9.1. Temporary halt for reasons of subject safety 
In accordance with section 10, subsection 4 of the WMO, the sponsor will suspend the study if there 
is sufficient ground that continuation of the study will jeopardize the health or safety of the subjects 
participating in the study. The sponsor will notify the accredited METc without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended pending a further 
positive decision by the accredited METc. The primary investigator will take care of the fact that all 
subjects are kept informed about the decisions.  
9.2. AEs, SAEs and SUSARs 
9.2.1. 
Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the 
study, whether considered related to the investigational product. All adverse events reported 
spontaneously by the subject or observed by the investigator, will be recorded. 
9.2.2. 
Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that 
• 
results in death; 
• 
is life threatening (at the time of the event); 
• 
requires hospitalization or prolongation of existing inpatients’ hospitalization; 
• 
results in persistent or significant disability or incapacity; 
• 
is a congenital anomaly or birth defect; 
• 
or any other important medical event that did not result in any of the outcomes listed above 
due to medical or surgical intervention but could have been based upon appropriate 
judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
The investigator will report the SAEs through the web portal ToetsingOnline to the accredited 
METc that approved the protocol, within 7 days of first knowledge for SAEs that result in death 
or are life threatening followed by a period of maximum of 8 days to complete the initial 
preliminary report. All other SAEs will be reported within a period of maximum 15 days after the 
sponsor has first knowledge of the serious adverse events. 
9.3. Follow-up of adverse events 
All AEs 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. SAEs need to be reported till end of 
study within the Netherlands, as defined in the protocol.  
 
 
 
 
 
 
 
 
 
 
 
 

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10. STATISTICAL ANALYSIS 
Statistical analyses will be performed using R. In general, continuous data will be presented as mean 
± standard deviation (SD) or median and interquartile range when skewed, and categorical data as 
counts and percentages. To correctly model the paired pre-post study design, we will use a linear 
mixed-effects model (lme4-package, version 1.1-35.2, Bates et al., 2015). This is performed by 
adding a patient-specific intercept and slope. As this is an exploratory stud, mean differences with a 
95% confidence interval will be considered as statistically significant. A false discovery rate (FDR) 
of 5% will be used in regression analyses, regarding SCFA and bacterial taxa, to correct for multiple 
testing. We will adjust for differences in age, sex, BMI, and fiber intake before the start of the 
intervention by including these as independent variables in the linear mixed-effects models described 
below. 
10.1. Primary study parameter(s) 
This study population consists of one group, where the primary endpoint is defined as the effect of 
WholeFiberTM for 6 weeks on fecal SCFA levels in surgically treated intermediate to high-risk 
cutaneous melanoma patients with no concurrent adjuvant treatment. The study is considered to 
have a positive outcome when paired differences are measured in fecal SCFA levels, comparing 
pre- and post-intervention. This will be analyzed using a linear mixed-effects model, modeling the 
fecal SCFA levels post-intervention as the dependent variable and fecal SCFA levels pre-
intervention together with patient-specific intercepts and slopes to take paired sampling into account. 
To further study the role of the gut microbiota, the relationship between post-intervention fecal SCFA 
levels and gut microbiota composition will be tested using a similar linear mixed-effects model. In 
this model, the post-intervention fecal SCFA levels will be modeled as the dependent variable and 
relative abundance of specific bacterial species together with patient-specific intercepts and slopes 
as independent variables.  
10.2. Secondary study parameter(s)  
To analyze changes in gut microbiota in surgically treated intermediate to high-risk cutaneous 
melanoma patients using WholeFiberTM for 6 weeks, we will use a similar linear mixed-effects model 
as described above.  In this model, the relative abundance of bacterial species post-intervention will 
be used as the dependent variable and their relative abundance pre-intervention together with 
patient-specific intercepts and slopes as independent variables. Furthermore, the effect of 
WholeFiberTM for 6 weeks on the microbial diversity will be evaluated using both alpha and beta 
diversity metrics. Alpha diversity metrics quantify within sample diversity and can be compared 
across groups. Beta diversity compares between sample diversity and is often calculated by 
computing a dissimilarity index (e.g. Bray-Curtis). 
10.3. Other study parameters 
Other study parameters such as anthropometry data or data from medical records will not be 
statistically tested but is used to describe the study population. 
 
 
 
 
 
 
 
 

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11. ETHICAL CONSIDERATIONS 
11.1. Regulation statement 
The study will be conducted in accordance with this protocol as well as the principles of the 
Declaration of Helsinki (64th WMA general assembly; October 2013), the ICH-GCP guidelines and 
other applicable laws and regulations, and in accordance with the principles of the Medical Research 
Involving Human Subjects Act (WMO). The study will start after approval of the medical ethical 
committee (METc) is obtained. The investigators in this study declare to have no conflict of interest. 
11.2. Recruitment and consent 
All participants are recruited from the UMCG. Also, patients who participated in earlier research 
projects may be re-invited. Patients who seem to meet the inclusion criteria are invited by their 
treating physician to consider participation. The researcher decides if the patient can make a well-
informed decision. The primary investigator provides information to the patient using the PIF and 
arranges a first inclusion visit with the patient (either on site or virtual). The patient is a given 2 weeks 
to consider participation. During this time, the patient is encouraged to council for advice using 
various sources. After one-week, additional questions can be answered and if the patient opts to 
participate the informed consent is signed. Documented informed consent must be obtained from all 
patients prior to inclusion in the study. Informed consent will be asked in writing, and in the following 
manner: the patient will sign and personal date the informed consent form first. When the investigator 
has signed and dated the consent form afterwards, the patient will receive a copy. Finally, the general 
physician of each patient will be informed about the enrolment of the patient to the study by a 
standard letter. The informed consent procedure takes place conform Good Clinical Practice (GCP). 
Documented informed consent must be obtained from all subjects prior to inclusion in the study. All 
subjects will be informed about their right to withdraw their agreement at any time without resulting 
in any claim of damage and about the possibility to ask for supplementary information from an 
independent physician. 
11.3. Benefits and risks assessment 
Participating in this study has a potential health benefit. It is known that an increased intake of fiber 
has a positive effect on the abundance of beneficial bacteria. In patients with melanoma, it is 
hypothesized that there’s an imbalance in the bacterial composition in the gut, leading to a reduction 
in beneficial bacteria. If melanoma patients experience a rise in beneficial bacteria, making the gut 
microbial composition more like the microbiome of healthy people, it may result in a more favorable 
disease outcome (reduced reoccurrence of the removed cutaneous melanoma). 
Participants could experience some bloating or flatulence, which are GI symptoms that are generally 
related to an increased fiber intake, when they start with WholeFiberTM. These discomforts are limited 
to the first few days of use and should not last longer than a few hours per day. In the previous 
clinical trials, using 30 g of the product, the participants did not report any major AEs in any of the 
study visits. The collection of feces and blood samples entails no risks for the subjects.  
 
 
 
 
 
 
 
 
 
 

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11.4. Compensation for injury 
The investigator has a liability insurance, which is in accordance with article 7, subsection 6 of the 
WMO. The sponsor(s) (also) has/have an insurance, which is in accordance with the legal 
requirements in the Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance 
for Clinical Research in Humans of 23rd June 2003). This insurance provides cover for damage to 
research subjects through injury or death caused by the study. 
 
1. € 450,000 (i.e. four hundred and fifty thousand Euro) for death or injury for each subject who 
participates in the Research; 
2. € 3,500,000 (i.e. three million five hundred thousand Euro) for death or injury for all subjects 
who participate in the Research; 
3. € 5,000,000 (i.e. five million Euro) for the total damage incurred by the organization for all 
damage disclosed by scientific research for the Sponsor as ‘verrichter’ in the meaning of said 
Act in each year of insurance coverage. 
 
The insurance applies to the damage that becomes apparent during the study or within 4 years after 
the end of the study. 
Note: an exemption for the insurance obligation, due to the negligible risk of this study is requested. 
11.5. Incentives  
Travel costs will be compensated: the subjects will receive a compensation of 0.21 euro/km when 
travelling by car. In addition, participants will receive a ticket for free parking at the UMCG on study 
days. The total amount of money will be paid after completing the study, and after the last samples 
have been collected. The dietary supplement will be given to the participant free of charge. 
Participating in the study will not result in costs for the participant.  
 
 

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1. Handling and storage of data and documents 
The handling of personal data complies with the Dutch ‘GDPR (General Data Protection Regulation) 
and AVG (Algemene Verordening Gegevensbescherming). All related documents and records will 
be kept and archived for at least 15 years. The collection and processing of participants’ personal 
information will be limited to what is necessary to ensure the study’s scientific practicability, the 
evaluation of efficacy, adherence, side effects and the investigational product’s safety. Information 
collected about participants during this clinical investigation will be treated confidentially. The 
investigator or her co- workers will collect data and transfer it without recording the patient’s name 
or date of birth. Instead, data will be coded with a participant identification number. This identification 
number consists of the study name and the participant number (e.g. MELFIB-001, MELFIB-002, 
etc.). 
The file with the key to the code will be managed by one person. The source documents will be kept 
in a locked file cabinet with limited access of the research personnel. In accordance with national 
laws and guidelines and the specifications of the ICH-GCP guidelines, the investigators are obligated 
to archive all documents pertaining to the study for the legally required period. 
The acquired data and examination results will be entered into an electronic case record form (eCRF) 
that is accessible via the internet. Investigators will receive personal usernames and passwords for 
this purpose, and data will be encrypted for transfer. It will be agreed before the start of the study 
which documents serve as source documents for all data included in the eCRF.  
12.2. Monitoring and quality assurance  
Associated investigators will be carefully selected and comprehensively informed and trained 
regarding Good Clinical Practice (GCP), all study procedures and the required examinations and 
documentation. 
The quality of data acquisition will be confirmed by regular monitoring visits as described in the 
Monitoring Plan. The monitor is independent from the study team and is not involved in the inclusion 
of participants and the design and implementation of the study. All monitoring activities will be in line 
with national laws and guidelines and the specifications of the ICH-GCP guidelines. 
According to the NFU Richtlijn Kwaliteitsborging Mensgebonden Onderzoek (versie September 
2023) this study can be identified as a low-risk study. Study monitors will visit the study site minimal 
once per year of which a minimal of two visits have to be on-site during the research period. Monitors 
will have access to all documents that are needed to perform their task according to the above-
mentioned guidelines. Monitors will check whether requirements to conduct the study are met and 
study procedures are followed correctly, and will check the study site’s documentation, the 
participants’ source data, eCRF entries, and the correct maintenance of the Investigator Site File. 
Investigators will permit trial-related monitoring, audits, ERB reviews and regulatory inspections, 
providing direct access to source data and study documents. 
 
 
 
 
 
 
 
 
 
 

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12.3. Amendments  
A 'substantial amendment' is defined as an amendment to the terms of the METc application, or to 
the protocol or any other supporting documentation, that is likely to affect to a significant degree:  
• 
the safety or physical or mental integrity of the subjects of the trial;  
• 
the scientific value of the trial;  
• 
the conduct or management of the trial; or  
• 
the quality or safety of any intervention used in the trial.  
All substantial amendments will be submitted for approval to the METc and to the competent 
authority. For non-substantial amendments, only a notification will be sent to the accredited METc, 
which will be recorded and filed by the investigator. Examples of non-substantial amendments are 
typing errors and administrative changes like changes in names, telephone numbers and other 
contact details of involved persons mentioned in the submitted study documentation. 
12.4. Annual progress report 
The investigator will submit a summary of the progress of the trial to the accredited METc once a 
year. Information will be provided on the date of inclusion of the first subject, numbers of subjects 
included and numbers of subjects that have completed the trial, serious adverse events/ serious 
adverse reactions, other problems, and amendments.  
12.5. Temporary halt and (prematurely) end of study report 
The sponsor will notify the accredited METc and the competent authority of the end of the study 
within a period of 90 days. The end of the study is defined as the last patient’s last visit. The 
investigator will notify the METc immediately of a temporary halt of the study, including the reason 
of such an action. In case the study is ended prematurely, the sponsor will notify the accredited 
METc and the competent authority within 15 days, including the reasons for the premature 
termination. Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, to the 
accredited METc and the Competent authority.  
12.6. Public disclosure and publication policy 
The sponsor of this study is the UCMG, where the main goal of the study is to contribute to research 
and to publish the results. The results of the study will be disclosed unreservedly, both positive and 
negative trial results must be disclosed. In general, the results of this research will be submitted for 
publication in an international peer-reviewed journal adhering to applicable privacy laws and 
regulations. Publication strategy will be determined by the principal investigator.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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13. STRUCTURED RISK ANALYSIS  
13.1. Potential issues of concern 
a. Level of knowledge about mechanism of action 
The gut microbiome composition of patients with an immunogenic tumor as an intermediate to high-
risk cutaneous melanoma (minimal stage II A) is currently unknown. However, a link between the 
gut microbiome, diet and response to immune checkpoint inhibitors in melanoma highlights the gut 
microbiome as a potential biomarker of response to treatment1. The gut microbiome can be modified 
through diet, and therefore is a potential therapeutic target.  
A higher intake of dietary fiber is linked to positive changes in the gut microbiota and an increase in 
SCFA-production35–39. Dietary fibers can’t be metabolized by human enzymes and therefore are 
fermented in the colon by the gut microbiota, which are able to produce SCFA. Daily intake of 15 g 
WholeFiberTM for 2 weeks has been shown to greatly modulate the gut microbiota and impact SCFA-
production in adults with prediabetes7. In our study, we aim to explore the effects of a WholeFiberTM 
intervention in surgically treated intermediate to high-risk cutaneous melanoma patients, with no 
concurrent treatment, on fecal SCFA levels and gut microbial composition. 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
WholeFiberTM has been tested before in a clinical trial in Dutch adults with prediabetes with 
positive effects, and a daily dosage of 30 g was well tolerated16. Currently, WholeFiberTM is under 
investigation in healthy adults with constipation complaints (HappyFiber study, NL80274.091.22, 
currently recruiting). 
c. Analysis of potential effect 
We expect that the WholeFiberTM intervention will greatly modulate the gut microbiota and increase 
fecal SCFA levels. The gut microbial composition and SCFA production are hypothesized to play an 
important role as potential therapeutic target for patients with an immunogenic tumor classified as 
an intermediate to high-risk cutaneous melanoma (minimal stage II A). However, this is an 
explorative study. If indeed we see these effects, this needs to be further tested in a large randomized 
controlled trial. 
d. Study population 
For this study, we will recruit around 20 surgically treated intermediate to high-risk cutaneous 
melanoma patients, with no concurrent adjuvant treatment. 
e. Interaction with other products 
WholeFiberTM is a dried vegetable that is safe for consumption and commercially available. We do 
not expect an interaction with other products as this is a food product.  
f. Predictability of effect 
In the previous study of Puhlmann et al. (2022) an increase of +26% in total SCFA levels was 
measured within two weeks after 15 g/day consumption of WholeFiberTM in adults with prediabetes. 
Furthermore, a 3-4-fold increase in butyrate-producing bacteria Anaerostipes and Bifidobacterium 
was observed. A similar effect can be expected in this study population. No previous studies have 
been performed before on the effects of WholeFiberTM in a population with cutaneous melanoma 
patients, however this study aims to explore these effects.  
g. Can effects be managed? 
Not applicable, WholeFiberTM is a dried vegetable, which is commercially available and proven safe 
for consumption. 
13.2. Synthesis 
All subjects included in this study are surgically treated intermediate to high-risk cutaneous 
melanoma patients, with no concurrent adjuvant treatment and no autoimmune disease. No children 
will be included in this study. Pregnancy is one of the exclusion criteria in this study.  
 

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14. REFERENCES 
1. Gacesa R, Kurilshikov A, Vich Vila A, et al. Environmental factors shaping the gut microbiome in a Dutch 
population. Nature. 2022;604(7907):732-739. doi:10.1038/s41586-022-04567-7 
2. Logan AC, Jacka FN, Prescott SL. Immune-Microbiota Interactions: Dysbiosis as a Global Health Issue. 
Curr Allergy Asthma Rep. 2016;16(2):13. doi:10.1007/s11882-015-0590-5 
3. Bach-Faig A, Berry EM, Lairon D, et al. Mediterranean diet pyramid today. Science and cultural updates. 
Public Health Nutr. 2011;14(12A):2274-2284. doi:10.1017/S1368980011002515 
4. Spencer CN, McQuade JL, Gopalakrishnan V, et al. Dietary fiber and probiotics influence the gut 
microbiome 
and 
melanoma 
immunotherapy 
response. 
Science. 
2021;374(6575):1632-1640. 
doi:10.1126/science.aaz7015 
5. Parada Venegas D, De La Fuente MK, Landskron G, et al. Short Chain Fatty Acids (SCFAs)-Mediated Gut 
Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Front Immunol. 
2019;10:277. doi:10.3389/fimmu.2019.00277 
6. Barrea L, Muscogiuri G, Frias-Toral E, et al. Nutrition and immune system: from the Mediterranean diet to 
dietary supplementary through the microbiota. Crit Rev Food Sci Nutr. 2021;61(18):3066-3090. 
doi:10.1080/10408398.2020.1792826 
7. Read S, Malmström V, Powrie F. Cytotoxic T Lymphocyte–associated Antigen 4 Plays an Essential Role in 
the Function of CD25ϩCD4ϩ Regulatory Cells that Control Intestinal Inﬂammation. Published online 2000. 
8. Annacker O, Asseman C, Read S, Powrie F. Interleukin-10 in the regulation of T cell-induced colitis. J 
Autoimmun. 2003;20(4):277-279. doi:10.1016/S0896-8411(03)00045-3 
9. Boets E, Gomand SV, Deroover L, et al. Systemic availability and metabolism of colonic‐derived short‐chain 
fatty acids in healthy subjects: 
a stable isotope study. 
J Physiol. 
2017;595(2):541-555. 
doi:10.1113/JP272613 
10. 
Tan J, Ribeiro RV, Barker C, et al. Functional profiling of gut microbial and immune responses toward 
different types of dietary fiber: a step toward personalized dietary interventions. Gut Microbes. 
2023;15(2):2274127. doi:10.1080/19490976.2023.2274127 
11. 
Hayday AC. γδ T Cell Update: Adaptate Orchestrators of Immune Surveillance. J Immunol. 
2019;203(2):311-320. doi:10.4049/jimmunol.1800934 
12. 
Li Y, Elmén L, Segota I, et al. Prebiotic-Induced Anti-tumor Immunity Attenuates Tumor Growth. Cell 
Rep. 2020;30(6):1753-1766.e6. doi:10.1016/j.celrep.2020.01.035 
13. 
Basu A, Ramamoorthi G, Albert G, et al. Differentiation and Regulation of TH Cells: A Balancing Act 
for Cancer Immunotherapy. Front Immunol. 2021;12:669474. doi:10.3389/fimmu.2021.669474 
14. 
Thomas AM, Fidelle M, Routy B, et al. Gut OncoMicrobiome Signatures (GOMS) as next-generation 
biomarkers for cancer immunotherapy. Nat Rev Clin Oncol. 2023;20(9):583-603. doi:10.1038/s41571-023-
00785-8 
15. 
Routy B, Lenehan JG, Miller WH, et al. Fecal microbiota transplantation plus anti-PD-1 immunotherapy 
in advanced melanoma: a phase I trial. Nat Med. 2023;29(8):2121-2132. doi:10.1038/s41591-023-02453-x 
16. 
Baruch EN, Youngster I, Ben-Betzalel G, et al. Fecal microbiota transplant promotes response in 
immunotherapy-refractory 
melanoma 
patients. 
Science. 
2021;371(6529):602-609. 
doi:10.1126/science.abb5920 

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Golonko A, Pienkowski T, Swislocka R, et al. Dietary factors and their influence on immunotherapy 
strategies in oncology: a comprehensive review. Cell Death Dis. 2024;15(4):254. doi:10.1038/s41419-024-
06641-6 
18. 
Puhlmann ML, Jokela R, Van Dongen KCW, et al. Dried chicory root improves bowel function, benefits 
intestinal microbial trophic chains and increases faecal and circulating short chain fatty acids in subjects at 
risk for type 2 diabetes. Gut Microbiome. 2022;3:e4. doi:10.1017/gmb.2022.4 
19. 
Passarelli A, Mannavola F, Stucci LS, Tucci M, Silvestris F. Immune system and melanoma biology: 
a balance between immunosurveillance and immune escape. Oncotarget. 2017;8(62):106132-106142. 
doi:10.18632/oncotarget.22190 
20. 
Reitmajer M, Leiter U, Nanz L, et al. Long-term survival of stage IV melanoma patients: evaluation on 
640 melanoma patients entering stage IV between 2014 and 2017. J Cancer Res Clin Oncol. 
2024;150(1):15. doi:10.1007/s00432-023-05533-0 
21. 
Reynolds A, Mann J, Cummings J, Winter N, Mete E, Te Morenga L. Carbohydrate quality and human 
health: a series of systematic reviews and meta-analyses. The Lancet. 2019;393(10170):434-445. 
doi:10.1016/S0140-6736(18)31809-9 
22. 
van Rossum C, Sanderman-Nawijn E, HAM Brants, Dinnissen C, Jansen-van der Vliet M. The Diet of 
the Dutch. Results of the Dutch National Food Consumption Survey 2019-2021 on Food Consumption and 
Evaluation with Dietary Guidelines. Rijksinstituut voor Volksgezondheid en Milieu RIVM; 2023. 
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Van Der Beek CM, Canfora EE, Lenaerts K, et al. Distal, not proximal, colonic acetate infusions 
promote fat oxidation and improve metabolic markers in overweight/obese men. Clin Sci. 
2016;130(22):2073-2082. doi:10.1042/CS20160263 
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Björk JR, Bolte LA, Maltez Thomas A, et al. Longitudinal gut microbiome changes in immune 
checkpoint blockade-treated advanced melanoma. Nat Med. 2024;30(3):785-796. doi:10.1038/s41591-
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25. 
Vich Vila A, Collij V, Sanna S, et al. Impact of commonly used drugs on the composition and metabolic 
function of the gut microbiota. Nat Commun. 2020;11(1):362. doi:10.1038/s41467-019-14177-z 
26. 
Zhao L, Zhang F, Ding X, et al. Gut bacteria selectively promoted by dietary fibers alleviate type 2 
diabetes. Science. 2018;359(6380):1151-1156. doi:10.1126/science.aao5774 
27. 
Lee KA, Thomas AM, Bolte LA, et al. Cross-cohort gut microbiome associations with immune 
checkpoint inhibitor response in advanced melanoma. Nat Med. 2022;28(3):535-544. doi:10.1038/s41591-
022-01695-5 
28. 
Bolte LA, Lee KA, Björk JR, et al. Association of a Mediterranean Diet With Outcomes for Patients 
Treated With Immune Checkpoint Blockade for Advanced Melanoma. JAMA Oncol. 2023;9(5):705. 
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29. 
Panagiotakos DB, Pitsavos C, Skoumas Y, Stefanadis C. The Association between Food Patterns and 
the Metabolic Syndrome Using Principal Components Analysis: The ATTICA Study. J Am Diet Assoc. 
2007;107(6):979-987. doi:10.1016/j.jada.2007.03.006 
 
 

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15. APENDIX 
15.1. Flow chart of study procedures 
This table describes the detailed measurements included per visit. An overview of the sampling 
timeline is depicted in figure 2.  
 
 
Screening Baseline 
Mid-trial 
(3 week) 
End-trial 
(6 week) 
Signed Informed Consent Forms1 
 
X 
 
 
Eligibility criteria 
X 
 
 
 
Medical, surgical, and cancer histories, 
including demographic information2 
 
X 
 
 
Clinical Evaluation/Anamnesis 3 
 
X 
X 
X 
Concomitant medication4 
X 
 
 
 
Weight and height5 
 
X 
 
 
Blood sampling6 
 
X 
X 
X 
Feces sampling7 
 
X 
X 
X 
FFQ 
 
X 
 
X 
HR-QoL 
 
X 
X 
X 
Bristol Stool Chart + GSRS 
 
X 
X 
X 
 
1Written informed consent can be obtained up to 30 days prior to study entry and is required for performing 
any study-specific tests or procedures. Results of standard-of-care tests or examinations performed prior to 
obtaining informed consent and within 28 days prior to study entry may be used for screening assessments 
rather than repeating such tests. The formal written consent for this trial of a subject must be obtained before 
initiation of any study-specific procedures. 
2Cancer history includes date of diagnosis. Demographic information includes sex, age, and self-reported 
race/ethnicity, smoking history.  
3CTCAE version 5.0 
4Concomitant medications include any prescription medications or over-the-counter medications, and 
nutritional supplements. At screening, any medications the subject has used within the 3 months prior to the 
baseline visit should be documented.  
5Weight and height will be assessed on the baseline visit. 
6Blood sampling of 10 ml whole blood to collect PBMCs and plasma for further analysis. 
7Feces sampling will be collected in a standardized manner as described in section 15.2 Feces sampling, 
participants will collect 3 frozen samples.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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15.2. Fecal sampling 
 
In deze instructie leest u welke materialen u van het onderzoeksteam ontvangt voor het 
verzamelen van ontlasting. In de beschrijving leest u stap voor stap hoe u de ontlasting thuis 
verzamelt. U wordt aangeraden de instructie eerst helemaal door te lezen voordat u daadwerkelijk 
de ontlasting gaat verzamelen.  
Heeft u vragen over het verzamelen van ontlasting? Neem dan gerust contact met ons op. 
U verzamelt de ontlasting thuis op één moment in de drie dagen voorafgaand aan de dag dat u 
een onderzoek afspraak heeft. Indien u bijvoorbeeld op woensdag een afspraak heeft; kunt u op 
zondag, maandag of dinsdag de ontlasting verzamelen. U vult alle buisjes in één keer.  
Het opvangen en bewaren van de ontlasting verloopt in zes stappen. 
• 
Opvangen van de ontlasting 
• 
Verzamelen van ontlasting in de pipet 
• 
Overbrengen van ontlasting in de pipet naar buisje 
• 
Verzamelen van ontlasting met gebruik van een schepje 
• 
Buisjes verzamelen in het zwarte zakje 
• 
Bewaren van het zwarte zakje met de buisjes in de vriezer 
In dit document worden de stappen nader toegelicht.  
Materialen 
Om de ontlasting te kunnen verzamelen ontvangt u de volgende materialen (controleer uw 
ontlasting verzamelpakket): 
• 
1x Grijs opvangbakje 
• 
1x Handschoenen 
• 
1x Ontlasting opvanger 
• 
1x Schaar 
• 
3x Buisjes 
• 
1 Buisje met schepje 
• 
3x Plastic pipet 
• 
1x Zwart zakje 
• 
1x Doorzichtig zakje 
• 
1x Koeltas 
• 
1x Ontlastingsverzamelformulier 
 
 
 
 
 
 
 
 

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Stap 1: Opvangen van de ontlasting 
Om ontlasting op te vangen, gebruikt u de meegeleverde ontlasting opvanger. 
• 
Vouw voorzichtig de ontlasting opvanger open in de richting van de pijltjes. 
• 
Bevestig deze met de plakstroken op het achterste deel van de wc-bril, zonder dat de 
plakstrip met het water in contact komt. 
• 
Vang de ontlasting op. 
 
Stap 2: Verzamelen van ontlasting in de pipet 
Na opvangen van de ontlasting, kunt u met de pipet de ontlasting verzamelen. Gebruik voor deze 
stap de handschoenen. 
• 
Knijp het ballonnetje bovenaan de pipet voorzichtig in en houd het uiteinde van de pipet in 
de ontlasting. 
• 
Druk de pipet tot ongeveer het eerste zwarte streepje in de ontlasting en laat het 
ballonnetje voorzichtig los. Door het loslaten van het ballonnetje kan de ontlasting een 
beetje omhoog gezogen worden, dat is niet erg. 
• 
Veeg de buitenkant van de pipet af met wc-papier. 
 
 
 
 
 
 
 

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Stap 3: Overbrengen van ontlasting in de pipet naar buisje 
Herhaal stap 2 en 3 zodat u in totaal 3 kleine buisjes met een pipet ontlasting heeft verzameld.  
• 
Breng het uiteinde van de pipet over in het kleine buisje met de schroefdop. Probeer hierbij 
geen contact te maken met de schroefdraad. 
• 
Knip het stukje pipet dat in het buisje zit af bij het tweede zwarte streepje.  
• 
Het stukje pipet wat overblijft kunt u weggooien in de prullenbak. 
   
 
 
 
 
 
 
 
Stap 4: Verzamelen ontlasting met gebruik van een schepje 
• 
Draai de dop met het schepje van de buis af. 
• 
Steek het schepje in de ontlasting. 
• 
 Schep de ontlasting op. 
• 
Breng het schepje naar het buisje en vul het buisje maximaal tot de helft. Probeer hierbij 
geen contact te maken met het schroefdraad van het buisje. 
 
 
 
Stap 5: Buisjes verzamelen in het zwarte zakje 

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• 
Doe de drie kleine buisjes en het buisje met schepje rechtop in het zwarte zakje. 
• 
Vul het ontlastingsverzamelingsformulier in en steek deze ook in het zwarte zakje. 
• 
Doe het zwarte zakje in het doorzichtige zakje, zorg ervoor dat uw gegevens zichtbaar 
blijven.  
 
Stap 6: Bewaar het zwarte zakje met de buisjes in de vriezer 
• 
Plaats de verzamelde materialen direct in de vriezer.  
• 
Bewaar het zwarte zakje in de vriezer tot de onderzoek afspraak, u neemt het zwarte zakje 
in de koeltas mee naar de afspraak. 
Het verzamelen van ontlasting is nu klaar en de ontlasting opvanger kan nu doorgespoeld worden. 
Wacht voor het doorspoelen een ogenblik, zodat het papier wat water opneemt en makkelijker 
door te spoelen is. Het grijze bakje kan in de afvalbak of container.