Pediatric Eosinophilic Esophagitis (pedEoE): Effect of Allergen Heat Denaturation on EoE Remission: a Pilot Trial
Phase: NA
Conditions: Eosinophilic Esophagitis
Sponsor: Universitaire Ziekenhuizen KU Leuven

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
 
Title: Pediatric Eosinophilic esophagitis (pedEoE): 
effect of  allergen heat denaturation on EoE 
remission: a pilot trial  
 
 
Investigators: 
Principal investigator:  
- 
Pediatric Allergy: prof dr Dominique Bullens 
 
Co-Investigator:  
- 
Pediatric Gastro-Enterology: prof dr Ilse Hoffman 
- 
Pathological Anatomy: prof dr Gert De Hertogh 
 
 
 
Signatures: 
 
Principal Investigator: 
 
 
Name: Dominique MA Bullens 
 
 
 
 
 
Date: 03-01-2023 
 
Signature 
 
 
 
 
Co-Investigators: 
 
 
Name:  Ilse Hoffman  
 
 
 
 
 
 
Date:  
 
Signature 
 
 
 
 
Name: Gert De Hertogh 
 
 
 
 
 
 
Date: 
 
Signature: 
 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
 
 
Content 
 
State of the art .........................................................................................................................................3 
Incidence………………………………………………………………………………………………………………………………………. 3 
Symptoms……………………………………………………………………………………………………………………………………… 3 
Diagnosis ……………………………………………………………………………………………………………………………………… 5 
Treatment  ............................................................................................................................................5 
Study objectives .................................................................................................................................... 12 
Primary end-point……………………………………………………………………………………………………………………..… 12 
Secondary end-points ........................................................................................................................ 12 
Material and methods ................................................................................................................. 13 
Statistical analysis………………………………………………………………………………………………………………………..….16 
Data policy…………………………………………………………………………………………………………………………………. 16 
Safety data…………………………………………………………………………………………………………………………………..19 
References…………………………………………………………………………………………………………………………………. 26 
 
 
 
 
 
 
 
 
 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
State of the art: 
 
Eosinophilic esophagitis is a chronic, antigen (food), driven immune-mediated disease 
characterized by : 1) symptoms of esophageal dysfunction , 2) histologic evidence of 
eosinophilic inflammation of the mucosa and is typically 3) affecting local esophageal tissue  
(1). It is a relatively newly defined disease for which pediatric guidelines were first described 
in 2014 (figure 1). 
 
The incidence of EoE is estimated to be 3,7/100.000/year and its prevalence is steadily 
increasing and current rates are similar as eg in a related disease inflammatory bowel disease 
with rates 1:1,000 (2). The disease shows a familial pattern in 2% of the subjects, raising the 
possibility of either a genetic predisposition (presentation in monozygotic twins 41% and in 
dizygotic twins 22%) or exposure to an unknown but common environmental factor (80%). In 
children is the average age between 6 and 10 years with a Male predominance (70% males) as 
well as Caucasian predominance. This disease is certainly associated with atopy as 26-50% of 
children with EoE (pedEoE) also have asthma; 30-90%  of them have allergic rhinitis, 19-55 
% have atopic dermatitis and 9-24 % have a well-defined IgE mediated food allergy (2). 
However, our own personal data retrospectively obtained in 81 children with pedEoE 
(s64441) indicates an even higher rate of associated IgE-mediated food allergy and IgE-
mediated comorbidities in over 80% of the children (PhD thesis Lisa Nuyttens-manama thesis 
Toon Dominicus). 
Presenting symptoms in children are different depending on the age of manifestation and are 
listed in figure 2A and 2B and Figure 3 (3, 4). Typical food impaction, as observed in adults 
with EoE, can be the presenting symptom in adolescents, but is seldom a symptom in young 
Figure 1 (1)  

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
children in whom vomiting, chronic nausea and feeding problems might be more frequent. 
Children develop spontaneous coping mechanisms to live with the disease, such as drinking a 
lot of fluids during meals, being unable to eat unless a drink is available, chew the food for a 
long time and/of cut the food in small pieces (3).  
 
 
Figure 2 A and B:  
Typical symptoms 
in children with 
EoE depending on 
age of presentation 
(3) 
A 
B 
Figure 3:  
Spread of typical 
symptoms in 
children with EoE 
depending on age 
of presentation (4) 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
The disease can be diagnosed if in presence of typical signs and symptoms, a gastro-
duodenoscopy with multiple biopsies in esophagus is performed. EoE can present 
macroscopically with  furrowing, white mucosal plaques, esophageal trachealization, 
esophageal narrowing, stricture, and mucosal tearing  (5).  EoE is suspected when at least 15 
eosinophils per high-powered field (hpf) (or 60 eos/mm2) are documented and non-EoE 
disorders that cause or potentially contribute to Esophageal Eosinophilia are ruled out (1, 5). 
Sensitivity of 100% and specificity of 96% can be obtained if the number of proximal and 
distal biopsies in the esophagus is increased to 2-4 biopsies proximal and distal. Therefore this 
number is currently recommended for successful diagnosis (5). Similarly, remission is defined 
as < 15 eosinophils per high-powered field and also requires 2-4 biopsies both in proximal 
and distal esophagus (5) 
Risk factors for the disease are represented in figure 4. 
Figure 4: Typical associated diseases in patients with EoE depicted from Gastroenterol 2018 (5) 
 
Unfortunately, EoE tends to become chronic and might lead to irreversible esophageal 
damage and esophageal strictures when untreated (6). 
Treatment is needed to improve the quality of life of patients with EoE, to stop the 
deposition of subepithelial fibrous tissue in the esophageal wall and to reduce the risk of 
severe esophageal injury by preventing long-lasting food impactions. Whether treatment goals 
should be focusing on improvement of symptoms, on reducing the esosinophilic inflammation 
and/or prevention of remodeling and reversal of fibrosis, is subject of intense debate. 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
Treatment guidelines now uniformly advise the use of high-dose proton-pump inhibitors (PPI) 
as first treatment line for 8 weeks, after which a new biopsy is recommended to search for 
remission (7). If the patient is in remission (eosinophils <5/hpf), long-term PPI (1 mg/kg/d) 
treatment is recommended and continued as long as yearly biopsies remain negative (and 
hence might be lifelong). On the other hand, if a patient is not yet in remission (eosinophils > 
15/hpf), or only has partial remission (eosinophils 5-15/hpf), a second treatment line is 
initiated. For this second treatment line, parents and children in accordance with their treating 
physician have two different options: 1) to start a treatment with local corticosteroids or 2) to 
start a treatment with food elimination diet.  
1) Option 1: use of local corticosteroids: 
a) Swallowed corticosteroids which are highly effective (response rate (RR) 
50-87 %):  Fluticasone (normally used as inhaler) (110 microgram , 2-4 
times/day) 
b) Budesonide (resolved in Xanthaan gum) <10 years : 1 mg; >10 years : 2 
mg 
c) (in adults orodispersible Budesonide Jorveza® has recently been approved) 
Patients are recommended to avoid drinking and eating for at least half an hour after 
administration; high RR but risk of side effects such as Infection : Candida albicans 
(10-15%), adrenal suppression, epithelial atrophy, growth delay (8). 
In UZ Leuven, if parents/children and treating physician agree on the use of option 1,  
treatment with local corticosteroids is initiated as Budesonide in Xanthaangum with 
subsequent biopsy to prove remission after 8 weeks treatment. If the patient is in 
remission, treatment is continued for at least a year till new biopsy, after which, if the 
patient remains in remission, sometimes a dose-reduction can be discussed. If the patient 
is not in remission, either an increase in the Budesonide dose or switch to option 2 will be 
discussed. Alternatively, the continuation of Budesonide with the start of a restriction diet 
in parallel can be discussed. 
 
2) Option 2: use of diet  (We recently published the remission rates for each of these 
diets based on meta-analysis (10).) 
a) Elementary diet (amino-acid (AA)) diet. First publication by Kelly et al in 
1995); RR in children 90%-95%; duration minimum 6 weeks; clinical 
improvement observed after 8,5  ± 3,8 days, histological remission in 2 
weeks (9). However, the diet is highly restrictive and almost impossible to 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
implement in older children even for a six week period. It moreover delays 
speech and has palatability issues (10). In real-life, the option of an 
elementary diet in Belgium after weaning is seldom discussed, as EoE 
unfortunately still forms no basis for reimbursement of the very expensive 
AA diet (although a reimbursement procedure has recently been 
started)(10). 
 
b) Empiric elimination of common dietary antigens. This diet is usually 
referred to as “6-food, 4-food, 2-food or 1-food” diet :  
6 food : cow’s milk, soy, eggs, wheat, nuts and fish; 4 food : cow’s milk, 
eggs, wheat, soy; 2 food : cow’s milk and wheat & 1 food : cow’s milk (9, 
10). A step-down or step-up approach for these empiric eliminations can be 
used. When a step-down treatment is chosen, the patient will first eliminate 
all foods, after which (if remission is obtained), new food products will be 
gradually introduced and 8 weeks after the introduction of each new food 
product, a gastroscopy with biopsy will be done to check for the 
maintenance of remission. If EoE re-occurs, the newly introduced food 
product is causal and will be permanently eliminated. If the step-up 
approach is chosen, a single food product is eliminated and if no remission 
is visualized on biopsy 8 weeks after elimination, an additional food 
product will be eliminated until remission is obtained and those foods will 
be eliminated permanently. More specifically: 
a. Step-down treatment starts as an 8 weeks 6-food diet after which a 
new biopsy is needed to prove remission. After this initial 
remission the diet is reduced with one specific food rationally 
chosen by the caregiver, the parents and the child for 8 weeks after 
which a new biopsy needs to prove whether or not the newly 
introduced food can stay in the diet or has to be removed again. 
This has to be repeated for each food product that will be 
reintroduced. As such, the diet starts with a high level of 
restrictions, has long diagnostic procedures and requires a high 
number of endoscopies. A new guideline tries to reduce the initial 
diet by starting from a 4 food diet instead, as nuts/peanuts and 
fish/shell-fish is only responsible for a limited number of cases (5) 

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b. Step-up treatment starts as an 8 week diet on cow’s milk alone after 
which a new biopsy is taken to study remission. If remission is 
obtained (RR 33% in children), the patients remains on a cow’s 
milk free diet. However, if there is no remission, a second food 
product needs to be eliminated. This in general might reduce the 
number of biopsies needed, but in children in whom after multiple 
weeks of limited and later-on very limited food product allowed, 
still no remission is obtained, the psychological barrier to add 
another food product to the forbidden list, which the chance of 
(again) failure to obtain remission, is an extreme burden.  
 
c) Allergy-driven Food Elimination 
Using allergy testing-guided food elimination in children results in a 
success rate in children that is slightly higher than the success rate of a 2-
food elimination diet, but is lower than its rate with a 4-food elimination 
diet  (2, 10). Its use is certainly more recommended in children when 
compared to adults in whom allergy-driven food elimination has a low 
success rate.  
In UZ Leuven if parents, children and treating physician agree on option 2 (diet-driven 
treatment), in infants and pre-school children it is adviced to start with a 1-food diet (milk) 
and if unsuccessful (after 8 weeks diet new biopsy shows no remission) a step-up protocol to a 
4-food diet (milk, eggs, wheat and soy) is adviced. As in Belgium soy intake in infants is 
usually limited, for the large majority of young infants and pre-school children this de facto 
means a 3-food diet (milk, eggs and wheat). In school-children and adolescents, children are 
advised to start with a 2-food or 4-food diet (depending on their willingness to adhere to the 
protocol) and to step-up to a 6-food diet immediately when after 8 weeks of initial diet no 
remission was obtained. Allergy testing usually occurs to rule out (associated) typical IgE 
mediated food allergy and/or pollen-associated risk factors with the chance of increased 
number of eosinophils in the esophagus during the pollen season and in this case, biopsies 
will not be taken during the pollen season.  
However, whereas in IgE-mediated pediatric food allergy recent evidence exists that heat-
denaturation of proteins might reduce their allergenicity and could lead to symptomfree 
introduction of certains recipes, nothing is known about the impact of heat denaturation on 
food proteins in pedEoE. Indeed, for several decades, it is known that food allergic 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
individuals tolerate heat-denaturated food proteins more easily than raw proteins. Moreover, 
the potential use of heat-denaturated food proteins to speed up tolerance towards allergens has 
recently been suggested. Hence, the hypothesis was generated that ingestion of small amounts 
of denaturated proteins might lead to higher rates of tolerance induction (11). We were able to 
demonstrate the change in food protein components upon heating of hen’s egg allergen in 
specific recipes, and furthermore reported that by gradual introduction of progressively less 
heated hen’s egg containing products, tolerance in infants was observed after 24-30 weeks in 
baked-egg tolerant subjects (12). In parallel, heat inactivation of cow’s milk proteins has also 
been reported (13) and results in gradual tolerance in cow’s milk allergic subjects (14). 
Moreover, the psychological impact of introduction of some heat denaturated food proteins in 
the diet of children with strict allergen avoidance, has been demonstrated to be of utmost 
importance (15). In pedEoE however, the effect of heat denaturation on the eosinophilic 
inflammation in the esophagus, has not yet been studied. We have several indications that 
allow us to hypothesize that heat denaturation in pedEoE could allow protein introduction 
while allowing remission rates, as observed in typical IgE-mediated food allergy. Indeed, 
pediatric EoE (pedEoE) (in contrast to EoE in adults) is linked in over 80% of cases to IgE 
mediated food and/or environmental allergy (PhD thesis Lisa Nuyttens, Manama thesis Toon 
Dominicus). Moreover, the same short list of 6 food products as  described in food allergy, 
namely milk, hen’s egg, wheat, soy, peanut/tree nuts, fish/shellfish is involved in more than 
60% of the pedEoE cases (10). In an almost similar percentage (58%) when compared to 6-
food diet, remission in pedEoE patients is possible using an allergy-test directed elimination 
diet (10) also pointing to involvement of IgE mediated allergy in pedEoE (2). Moreover, 
allergen oral immunotherapy protocols in IgE mediated food allergic patients on the opposite, 
can induce pedEoE in small numbers between 0.5-1% (16). We furthermore demonstrated in a 
patient with eosinophilic gastro-enteritis that plant-food pollen-associated eosinophilic 
increase can be the driving force of the disease (17). All of these point to the involvement of 
IgE mediated allergy in pedEoE opposed to EoE in adults. Finally, recently a report in a boy 
with cow’s milk protein induced EoE, demonstrated that heat-denaturation of the cow’s milk 
proteins induced remission, in a cow’s milk driven pedEoE (18). We therefore will study, 
similarly as in children with IgE mediated food allergy, the possibility to introduce heat-
denaturated cow’s milk or hen’s egg protein instead of complete avoidance in order to induce 
remission in pedEoE. To that aim, we intend to provoke 8 pedEoE children on a hen’s egg 
and/or cow’s milk free diet, with heat denaturated hen’s egg and/or cow’s milk proteins and 
plan a biopsy to study remission after 8 weeks of introduction at home. If the (re)introduction 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
was succesfull, we will introduce recipes with gradually less heated proteins after which 
sequentially a new biopsy will be performed after 8 weeks of introduction. Although it can be 
anticipated that introduction of the least heated step (raw egg and/or uncooked (UHT-)treated 
cow’s milk) will induce recidive of the disease, we will discuss with parents whether or not 
they would accept an 8-weeks reintroduction protocol of the final step and afterwards 
observation of its effect on the esophagus in their children. One might indeed even hope for 
tolerance induction while using this graded protocol, similar as in food protein allergic 
subjects, although we have no indication of such a tolerance induction in EoE patients, who 
today are taught to avoid these allergens “live-long”. 
In contrast to the results related to allergen sIgE, allergen-specific IgG4 (sIgG4) is more 
consistently associated with oral tolerance (18bis). Allergen sIgG4 antibodies can compete 
with IgE for allergen binding to type II IgE receptor-expressing cells, such as basophils and 
mast cells, leading to the blockage of this receptor. The production of both IgE and IgG4 
antibodies is known to be up-regulated by interleukin-4 (IL-4) produced from activated Th2 
cells. However, IL-10 secreted by Treg cells can suppress IgE production and simultaneously 
increase IgG4 production (Figure 5). Therefore, IgE and IgG4 production are not always 
similarly increased. Not only are IgE and IgG4 important to predict reactivity to an allergen or 
to confirm food tolerance, also the ratio of IgE to IgG4 seems to play a key role (19). 
Okamoto et al. studied  the concentrations of hen’s egg sIgE and IgG4 antibodies during oral 
food challenges (OFC) with egg white in children sensitized to hen’s egg. Hen’s egg sIgE 
concentrations were higher in children with a positive OFC than in children with a negative 
OFC. On the other hand, the concentration of hen’s egg sIgG4 antibodies was higher in the 
negative OFC than in the positive food challenge. However, the ratio of IgE to IgG4 seemed 
to be an even more useful parameter than IgE or IgG4 alone to predict clinical reactivity 
during hen’s egg challenge as it also had a more accurate sensitivity and specificity (20, 21). 
Similarly for cow’s milk, Savilahti et al. showed higher levels of cow’s milk sIgE in cow’s 
milk allergic children compared with those who became tolerant by the age of 3 years, but 
cow’s milk sIgG4 serum concentrations were lower among patients with persistent cow’s 
milk allergy than among patients tolerating cow’s milk by the age of 3 years (20, 21). 
Regulatory B (Breg) cells are immunosuppressive B cells, characterized by the production of  
IL-10, IL-35 and transforming growth factor beta (TGF-β). IL-10 secreting Breg (Br1) cells, 
are known for being the main producers of IgG4 (19). Through the secretion of IL-10, IL-35 
and TGF-β, Breg cells are known to suppress the activation and proliferation of effector T 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
cells, and also induce the differentiation of Foxp3+ regulatory T cells and type 1 regulatory 
(Tr1) cells (Figure 5). 
Some indication exist that systemic as well as local (in the esophagal lesion) food specific 
IgG4 could be linked to the EoE as well (22). In that sense, we might predict that if our 
hypothesis stands, serum food specific IgG4 could be used as biomarker for the disease 
(and/or for tolerance towards a certain food protein). Moreover, in vitro activation of PBMC 
from those pedEoE children by (gradually less heat-denaturated) food proteins (+/- the B cell 
activating cytokine IL-21 (23)) and afterwards flow cytometric B cell staining with B (and 
plasma) cell activation markers, could be developped as an in vitro diagnostic test to search 
for B cell activation in those children. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 5: Figure by Nuyttens L et al, in revision (19) 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
Study objectives: 
The objective of the study is to study whether the introduction of heated food products (more 
specifically heated hen’s egg and/or cow’s milk) in children with EoE would be possible 
without re-occurrence of the eosinophilic inflammation, while the intake of less heated 
products might cause disease recidive.  Moreover, we would like to study whether the gradual 
re-introduction of less heated products after the most heated form is tolerated, could lead to 
tolerance induction in EoE. 
Primary end-point:  
- 
Primary end-point of the study is to study the number of ped EoE children by 
inclusion on hen’s egg and/or cow’s milk free diet with potential remaining remission 
(<15 eosinophils/hpf on esophagal biopsies) after 8 weeks (re)introduction of heat-
denaturated hen’s egg and/or cow’s milk proteins (introduced sequentially as on the 
one hand baked egg (cake), hard-boiled egg, omelet and soft-boiled egg and on the 
other hand 20’ cooked cow’s milk, 15’, 10’ and 5’ cooked cow’s milk). 
 
Secondary end-points: 
- 
The number of pedEoE children in whom remission remain with introduction of cake 
and/or 20’cooked cow’s milk (step 1), but no longer with less heated proteins 
- 
To study whether remission is associated with specific circulating and/or local food 
protein IgG4 reduction (when baseline levels (serum) would be (stored) available) 
(and increase upon unsuccessful introduction of less heated proteins) 
- 
To study whether remission could be associated with changes in local IgG4 production 
(on biopsy staining) 
- 
To study whether we could develop an in vitro mimicking B cell activation test by 
stimulating PBMC with food proteins obtained after different heat-denaturating steps 
- 
Can we observe a correlation between Pediatric EoE PEESS v2.0 and Peds-QL scores  
(obtained in all ped EoEchildren participating in study s64441) and histologic 
remission? 
- 
To study the presence of associated atopy (tree-, grass- or weed pollen or fungi 
allergy)  
- 
To study the presence of associated typical IgE mediated food allergy (sensitization 
and documented symptoms) 
 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
 
Material and methods 
- 
Inclusion criterion: All children (aged 12 months and older) presenting (since 1-1-
2014: moment of diagnostic guideline standardization for pedEoE) with diagnosed 
pedEoE at UZ Leuven eliminating either hen’s egg or cow’s milk or both and in 
complete remission after their latest biopsy (no longer than 12 months earlier, but 
preferentially as short as possible after their latest biopsy) are eligible for the study. If 
the last biopsy has been performed more than 12 months earlier, a new gastro-
duodenoscopy will be performed to verify remission and rule out e.g. active gastritis. 
If they are on PPI and/or local budesonide treatment, this should be stable for at least 3 
months and will remain untouched during the entire study. We will include 18 pedEoE 
subjects in total. 
- 
Exclusion criteria:  
- 
Children younger than 12 months 
- 
Children with active pedEoE 
- 
Children who refuse to adhere to the protocol 
- 
Children with associated IgE mediated hen’s egg and/or cow’s milk allergy 
with specific IgE antibody titers that predict active (baked egg and/or baked 
milk) food allergy with cut-off titers as used at the consultation of allergy 
(KLL).  Those children will become eligible however if their titers decrease 
while the study is still open. 
 
 
 
Study design: 
Study subjects and study procedure 
If inclusion criteria are met, after obtaining informed consent from the parents (and when old 
enough, assent from the children) 18 children will be included in this pilot pedEoE trial. 
Children will be provoked in-house (UZ Leuven petit-cru), based on the written procedure 
available for heated egg (wheat or rice flour based) or cow’s milk provocation. If on diet for 
both, parents and children will decide which allergen they will start with, but both food 
products will be introduced sequentially in time (this is after complete termination of the first 
food introduction). Afterwards, they will introduce heated egg by cake or 20’ boiled cow’s 
milk at least three times a week for 8 weeks (as for the TETI study (10)) and will return to the 
hospital for a gastroscopy with biopsies. EoE remission will be studied on 2X3 biopsies 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
(upper and lower esophagus) by eosinophil staining within the routine anatomopathological 
laboratory within our hospital. 
If remission remains, subjects will be invited to introduce 10’ hard-boiled egg (consecutively 
hard-boiled egg white; hard-boiled egg yolk) or 15’boiled cow’s milk at home (2-3 times a 
week) for 8 weeks, after which they will return to the hospital for the second gastroscopy with 
similar procedure.  
If remission is lost, they will return to the strict avoidance diet for at least 10 weeks (allowing 
2 weeks of additional healing) after which they will return to the hospital for their second 
gastroscopy. Upon remission after avoidance, the children will remain eligible for a second 
heat denaturated food challenge (when on diet for both). When they are not on diet for both 
hen’s egg and cow’s milk, the study for them will end (final visit). 
In children in whom remission remained after introduction of either hard-boiled egg or 
15’boiled cow’s milk, regular intake of 7’ baked omelet or equivalent (waffle, pancake) or 
10’boiled cow’s milk will be introduced for 8 weeks after which they will return to the 
hospital for gastroscopy. Similarly as above, subjects will return to complete avoidance or 
(upon parental decision in agreement with treating physicians) they can return to introduction 
of the tolerated step (step 1) for 10 weeks, after which a new gastroscopy will be performed. 
When they are not on diet for both hen’s egg and cow’s milk, the study for them will end 
(final visit).  
Similarly as above introduction of step 4, 4’ soft-boiled egg or 5’ boiled cow’s milk will be 
introduced at home for 8 weeks afterwards upon remaining remission. Gastroscopy with 
biopsies is planned afterwards. If introduction of step 3 (omelet or 10’ boiled cow’s milk) was 
no longer associated with remission, children will return to complete avoidance or (upon 
parental decision together with treating physician) to step 1 or step 1+2.  
Children who were able to tolerate step 4 for either hen’s egg or cow’s milk, will then be 
offered the possibility to choose for either termination of the study or (optionally) introduce 
either raw egg (tiramisu or chocolate mousse) or uncooked UHT-treated cow’s milk for 8 
weeks after which gastroscopy with biopsies will be performed. 
For all steps if symptoms would (re)occur, gastroscopy with biopsy will be planned earlier 
and the introduction of the step will be on hold, until the biopsy would have proven remaining 
of remission. Upon EoE recidive, the patient will return to complete avoidance. 
 
Number of study visits (n=9+4 telephone visits): 
T-1 
V0: screening visit: within SOC; informed consent and assent 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
T0 
V1: visit 1: step 1 in hospital provocation and biomarker 
T8   
V2: 8 weeks later visit 2: gastroscopy with biopsy and biomarker 
V2T: telephone guidance discussing biopsy results (Go/no go V3; if no go: V8 and V9 
termination visit) 
T9 
V3: visit 3: clinical examination and dietary advice step 2 
T17 
V4: 8 weeks later visit 4: gastroscopy with biopsy and biomarker 
V4T: telephone guidance discussing biopsy results (Go/no go V5; if no go V8 and V9 
termination visit) 
T18 
V5: visit 5: clinical examination and dietary advice step 3 
T26 
V6: 8 weeks later visit 6: gastroscopy with biopsy and biomarker 
V6T: telephone guidance discussing biopsy results 
T27 
V7: visit 7: clinical examination and dietary advice step 4 
T35 
V8: 8 weeks later visit 8: gastroscopy with biopsy and biomarker 
V8T: telephone guidance discussing biopsy results 
T37 
V9: visit 9: termination visit 
T45 
V10 optional: new biopsy after step 5 (unlimited, unheated egg or UHT milk) 
After at least 6 weeks post V9 (or V10 if applicable), inclusion for the second food allergen 
can occur (cross-over). 
 
Blood and esophagus biomarker study 
 
IgG4 staining on the biopsies will be performed by prof de Hertogh. Baseline blood (2X10 
mL) will be obtained before the in-house provocation procedure and serum will be stored for 
specific IgG4 analysis later on. In parallel, PBMC will be isolated at the Allergy and Clinical 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
immunology Research Laboratory (LACI) and stimulated in vitro with heat denaturated 
proteins, as routinely performed within LACI. B (and plasma) cells will be stained by 4-
laserflow cytometry (BD Fortessa). IgG4 production in supernatants will be studied by 
ELISA (specific IgG4 by Immunocap 100) and membrane bound IgG(4) (on biopsy material) 
by flow cytometry. This procedure will be repeated during each gastroscopy (when IV line is 
placed). 
Parents will be offered a parking ticket for the visits in UZ Leuven and a lunch ticket during 
the day of the study visits with gastroscopy. After the study is finished, study participants (and 
their parents) will be offered a ticket for an attraction park (choice out of 4 parks). 
 
Statistical analysis 
The study is part of a funded FWO-TBM project that will start in October 2023. We will start 
the inclusion of the study trial from July 2023 on using internal funding for the first inclusion 
when needed. We hypothesize that the majority of children will start with hen’s egg 
introduction, given the fact that more recipes containing hen’s egg are existing and might 
change the lives of parents and children drastically when allowed. We hence do not anticipate 
or intend equal distribution of hen’s egg and cow’s milk inclusion.  If in all 18 children step 1 
induces reoccurrence of the disease, the study will be stopped and no cross-over to the second 
food allergen will any longer be possible. 
Data reporting will be mainly descriptive. No statistical testing is foreseen. 
 
Data policy  
Ethics and regulatory approvals 
The Participating Site and Investigator will perform the Study fully in accordance with the 
terms of the Protocol, this Agreement including all its appendices and Applicable Laws and 
Regulations (as defined as follows: the current version of the World Medical Association’s 
Declaration of Helsinki, the guidelines and guidance documents specifying Good Clinical 
Practice (“GCP”) and guidelines of competent authorities; all applicable laws, rules and 
legislation in relation to clinical trials, use of human biological material, data protection and 
the processing of personal data, and patient’s rights, including but not limited to the General 
Data Protection Regulation 2016/679 (“GDPR”) and the Belgian law of 30 July 2018 on the 
protection of natural persons with regard to the processing of personal data). The Participating 
Site will ensure that the Investigator and all employees, agents, contractors and/or the 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
representatives  involved in the performance of the Study (jointly “Staff”) are properly trained 
and sufficiently qualified. The Participating Site shall provide Staff with all necessary 
documentation to allow the proper performance of the Study, which shall be conducted in 
compliance with the Protocol. The Participating Site shall be responsible to ensure that all 
work performed by Staff is done in compliance with the Protocol, this Agreement and all 
Applicable laws and Regulations. The Participating Site undertakes to impose on all 
employees, agents, contractors and/ or representatives the terms and conditions not less strict 
than those set out in this Agreement. 
 
Insurance/Indemnity  
In accordance with the Belgian law relating to experiments in humans dated May 7, 2004, 
and/or with the Belgian law relating to clinical trials dated May 7, 2017 (as applicable) 
Sponsor shall be liable, even without fault for any damages incurred by the Study Subject and 
linked directly or indirectly to the participation to the Study, and shall provide compensation 
therefore through its insurance program.  
Before commencing the Study, the Sponsor shall enter into an insurance contract which 
covers this liability, and the liability of every individual intervening in the Study, irrespective 
of the nature of the affiliation between the intervening individual, the Sponsor and the Study 
Subject. Every contractual provision aiming at limiting this liability is considered null and 
void. 
Investigator and Sponsor shall have and maintain in full force and effect during the term of 
this Agreement (and following termination or completion of the Study to cover any claims 
arising from the Study) adequate insurance coverage for: (i) medical professional and/or 
medical malpractice liability, and (ii) general liability resulting from the Study at the 
Participating Site required by Belgian law 
 
 
Confidentiality and privacy 
Data will be stored for 25 years on a password-access limited and secured UZ Leuven server. 
The pseudonymization key will be stored separately from the data and only be accessible for 
the principal investigators of the study. The Sponsor, Participating Site and Investigators shall 
treat all information and data relating to the Study as confidential and with respect to the 
Participant’s privacy.  The Sponsor, Participating Site and Investigators shall not disclose 

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Heat denaturation in PedEoE-pilot-version 5 July 2023 
 
such information to any third parties or use such information for any purpose other than the 
performance of the Study.  
 
Publication Policy 
It is anticipated that the results of the overall Study shall be published in a publication, 
involving the data of all participants in the Study. 
Publications will be coordinated by the Investigator of Sponsor. Authorship to publications 
will be determined in accordance with the requirements published by the International 
Committee of Medical Journal Editors and in accordance with the requirements of the 
respective medical jour 
 
 

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Adverse Event Reporting 
1. 
Definitions 
1.1. Adverse Event (AE) 
An AE is any untoward medical occurrence in a patient or subject during an 
experiment, and which does not necessarily have a causal relationship with this 
treatment. 
An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the use of a 
product, whether or not considered related to the product. Any worsening (i.e., any 
clinically significant adverse change in the frequency or intensity of a pre-existing 
condition) should be considered an AE. 
1.2 
Adverse Reaction (AR) 
An AR is any untoward and unintended responses to an investigational medicinal 
product or to an experiment and, when an investigational product is concerned, 
related to any dose administered. 
1.3 
Serious Adverse Event (SAE) 
An SAE is any untoward medical occurrence that results in any of the following: 
• 
Death 
• 
A life-threateninga experience 
• 
In-patient hospitalisation or prolongation of existing hospitalisation 
• 
A persistent or significant disability or incapacity 
• 
A congenital anomaly or birth defect 
• 
Important medical events that may be considered an SAE when - based on 
appropriate medical judgement - they may jeopardise the subject and may require 
medical or surgical intervention to prevent one of the above outcomes 
 
a The term “life threatening” in the definition of SAE refers to an event in which the subject was at risk 
of death at the time of the event. It does not refer to an event which hypothetically might have caused 
death if it was more severe. 
 

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1.4 
Adverse Events of Special Interest (AESI) 
  Define here any AEs that are of special interest (e.g. specific for the intervention) and 
must follow the same reporting timelines as SAEs. 
The following events should be reported within the same timelines as SAEs: 
- 
none 
 
2 
Adverse Events that do not require reporting 
   Define here any AEs or SAEs that are expected and do not require reporting for this 
trial. The following should not be reported as AEs: 
• 
Pre-existing conditions, including those found as a result of screening (these 
should be reported as medical history or concomitant illness). 
• 
Pre-planned procedures unless the condition for which the procedure was 
planned has worsened from the first trial-related activity after the subject has signed 
the informed consent. 
 
The following events are commonly observed in the study population and are 
therefore not considered  as adverse events for the purpose of the trial: 
• (seasonal) allergic rhinoconjunctivitis, (seasonal) asthma attacks, atopic dermatitis  
Although these events should not be reported to the Sponsor, these should be 
recorded in the patient’s medical notes according to routine practice.  
The following events not to be considered as SAEs are: 
• 
Pre-planned hospitalisations unless the condition for which the hospitalisation 
was planned has worsened from the first trial-related activity after the subject has 
signed the informed consent.  
• 
Hospitalisation as part of a standard procedure for protocol therapy 
administration. However, hospitalisation or prolonged hospitalisation for a 
complication of therapy administration will be reported as an SAE. 
• 
Hospitalisation or prolongation of hospitalisation for technical, practical, or 
social reasons, in absence of an AE. 
 
3 
Recording and reporting of Adverse Events 

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   See also: Safety Reporting ; Documents. 
The information listed below that should be recorded for each AE can be 
supplemented with additional information. These data points should be incorporated 
in the (e)CRF. 
Specific timelines for reporting AEs in the (e)CRF can be added, e.g. within 7 days after 
becoming aware. 
Investigators will seek information on AEs during each patient contact. All events, 
whether reported by the patient or noted by trial staff, will be recorded in the 
patient’s medical record and in the (e)CRF within a reasonable time after becoming 
aware. If available, the diagnosis should be reported on the AE page, rather than the 
individual signs or symptoms. If no diagnosis is available, the Investigator should 
record each sign and symptom as individual AEs. 
The following minimum information should be recorded for each AE: 
- AE description 
- start and stop date of the AE 
- severity 
- seriousness 
- causality assessment to the study interventions 
- outcome 
 
3.1 
Assessment  
   If specific assessment references are to be used for events reported in the trial, this 
should be detailed in the below section. The below guidances can be modified 
according to the specifications of the trial. 
All AEs must be evaluated by an Investigator as to: 
 
• 
Seriousness: whether the AE is an SAE. See above for the seriousness criteria. 
• 
Severity: Severity must be evaluated by an Investigator according to the 
following definitions: 
⎯  
Mild – no or transient symptoms, no interference with the subject’s 
daily activities 

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⎯  
Moderate – marked symptoms, moderate interference with the 
subject’s daily activities 
⎯  
Severe – considerable interference with the subject’s daily activities, 
unacceptable 
• 
Causality: 
⎯  
None – An AE which is not related to the study-related interventions 
⎯  
Unlikely – An AE for which an alternative explanation is more likely (e.g. 
concomitant medication(s), concomitant disease(s)), and/or the relationship in 
time suggests that a causal relationship is unlikely 
⎯  
Possible – An AE which might be due to the study-related interventions. 
An alternative explanation is inconclusive. The relationship in time is 
reasonable; therefore the causal relationship cannot be ruled out. 
⎯  
Probable - An AE which might be due to the study-related intervention. 
The relationship in time is suggestive (e.g. confirmed by dechallenge). An 
alternative explanation is less likely. 
⎯  
Definitely – An AE which is known as a possible adverse reaction and 
cannot be reasonably explained by an alternative explanation. The relationship 
in time is very suggestive (e.g. it is confirmed by dechallenge and rechallenge). 
 
3.2 
Timelines for reporting 
   The below timelines for adverse event reporting are to be made study-specific. The 
safety reporting flow should be described in detail in the below section (eg. through 
eCRF or paper forms). 
- 
After informed consent has been obtained and after initiation of study-related 
interventions:  
o 
All AEs, SAEs and AESIs causally related to a study-related intervention 
will be reported until 30 days after the last study-related intervention or until 
last follow-up visit (whichever occurs first). 
All SAEs and AESI as defined in the protocol must be reported to the Sponsor within 24 
hours of the trial staff becoming aware of the event. The immediate report shall be 

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followed by detailed, written reports. The immediate and follow-up reports shall 
identify subjects by Trial identification. 
 
SAE details will be reported by the Investigator to the Sponsor:  
• 
By completing the SAE form in the (e)CRF OR  
If an authorised Investigator from the reporting site is unavailable, initial reports 
without causality and expectedness assessment should be submitted to the Sponsor 
by a healthcare professional within 24 hours of becoming aware of the SAE, but must 
be followed-up by medical assessment as soon as possible thereafter. 
 
3.3 
Follow-up  
The Investigator must record follow-up information by updating the medical records 
and the appropriate forms in the (e)CRF. The worst case severity and seriousness of an 
event must be kept throughout the trial. 
SAE follow-up information should only include new (e.g. corrections or additional) 
information and must be reported within 24 hours of the Investigator’s first 
knowledge of the information. This is also the case for previously non-serious AEs 
which subsequently become SAEs. 
• 
All SAEs must be followed up until the outcome of the event is ‘recovered’, 
‘recovered with sequelae’, ‘not recovered’ (in case of death due to another cause) or 
‘death’ (due to the SAE) and until all related queries have been resolved, or until end 
of trial (whichever occurs first). 
• 
Non-serious AEs must be followed up until the patient’s last study visit, and 
until all related queries have been resolved. 
 
SAEs after the end of the trial: If the Investigator becomes aware of an SAE with 
suspected causal relationship to the study-related interventions after the subject has 
ended the trial, the Investigator should report this SAE within the same timelines as 
for SAEs during the trial. 
 
3.4 
Pregnancy 

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Female subjects must be instructed to notify the Investigator immediately if they 
become pregnant during the trial. The Investigator must report any pregnancy in 
subjects during the trial to the Sponsor. 
 
3.5 
Death 
All deaths will be reported without delay to the Sponsor (irrespective of whether the 
death is related to disease progression, study procedure or is an unrelated event). The 
sponsor will notify all deaths, as soon as possible after becoming aware, to the Central 
EC and the EC of the concerned site and provide additional information if requested. 
 
4 
Reporting requirements to Ethics Committee’s (EC’s) 
The Investigator is responsible for ensuring that all safety events are recorded in the 
(e)CRF and reported to the Sponsor in accordance with instructions provided below. 
The Sponsor will promptly evaluate all SAEs and AESIs against medical experience to 
identify and expeditiously communicate possible new safety findings to Investigators 
and based on applicable legislation. 
 
4.1 
Annual reporting 
The sponsor has the obligation to, once a year throughout the clinical trial (or on 
request), submit a progress report to the EC’s containing an overview of all SARs 
occurred during the reporting period and taking into account all new available safety 
information received during the reporting period. 
 
 
 
 
 
 
 
 

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4.2 
Overview reporting requirements 
 
WHAT 
HOW 
TO 
TIMELINES 
Investigator 
AE 
AE form 
sponsor 
as defined in protocol 
SAE 
SAE form 
sponsor 
Immediately (within 24 
hours 
of 
becoming 
aware of the event) 
Exceptions: as defined in 
protocol 
death 
SAE form 
sponsor 
asap 
Sponsor 
death 
SAE form + 
narrative 
Ethics Committees 
asap 
Annual Progress 
Report 
APR template 
Ethics Committees 
annually 
 
 
5 
Pharmacovigilance / Materiovigilance 
   The below section is applicable for experiments where a medicinal product will be 
used by the participants according to the standard of care. 
Any adverse reactions related to a medicinal product used by the participants in the 
study, can be reported to the Compentent Authorities according to the 
pharmacovigilance guidelines. 
 
 
 

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