Safety and Feasibility of the Use of Cryoablation in Patients With Brain Tumors
Phase: NA
Conditions: Brain Neoplasm, Brain Metastases, Glioma, Meningioma
Sponsor: Erasmus Medical Center

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RESEARCH PROTOCOL 
 
Safety and feasibility of the use of cryoablation 
in patients with brain tumors 
 
Erasmus MC, Department of Neurosurgery 
 
 
 

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PROTOCOL TITLE  
Safety and feasibility of the use of cryoablation in patients with brain neoplasm 
Protocol ID 
NL81429.078.22 
(PaNaMa-nummer: 9358) 
Short title 
Cryoablation of brain neoplasm 
EudraCT number  
NA 
Version 
0.6 
Date 
January 5th, 2024 
Project leader 
 
Prof. Dr.  A.J.P.E. Vincent M.D, PhD 
Erasmus University Medical Center 
Department of Neurosurgery 
Doctor Molewaterplein 40 
3015 GD Rotterdam 
The Netherlands 
0031-10-7040704 
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
 
Co-investigator 
Prof. Dr. A.J.P.E. Vincent M.D. PhD 
 
 
 
S. Mohammadian 
Erasmus University Medical Center 
Department of neurosurgery 
Doctor Molewaterplein 40 
3015 GD Rotterdam 
The Netherlands 
0031-10-7036467 
 

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Sponsor (in Dutch: 
verrichter/opdrachtgever) 
Erasmus MC Rotterdam 
 
 
Subsidising party 
Erasmus MC Rotterdam 
Doctor Molewaterplein 40, 3015GD Rotterdam 
0031-10-7040704 
Independent expert (s) 
Dr. M.L.C.van Veelen neurosurgeon, Head of 
Department of Pediatric  Neurosurgery Sophia/ 
Children’s Hospial, Erasmus Medical Center 
Rotterdam 
The Netherlands 
010-7035993 
Laboratory sites  
NA 
Pharmacy  
NA 
 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Head of Department: 
Prof. dr. C.M.F. Dirven M.D. 
PhD, neurosurgeon 
Head of Department of 
Neurosurgery 
 
05-01-
2024 
[Coordinating 
Investigator/Project 
leader/Principal Investigator]:  
Prof. Dr. A.J.P.E. Vincent M.D. 
PhD, neurosurgeon, 
Department of Neurosurgery 
 
05-01-
2024 
 
 

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TABLE OF CONTENTS 
 
 
1. 
INTRODUCTION AND RATIONALE ............................................................................... 10 
2. 
OBJECTIVES .................................................................................................................. 12 
3. 
STUDY DESIGN ............................................................................................................. 13 
4. STUDY POPULATION ....................................................................................................... 15 
4.1 Population (base) .......................................................................................................... 15 
4.2 Inclusion criteria ............................................................................................................ 15 
4.3 Exclusion criteria ........................................................................................................... 15 
4.4 Sample size calculation ................................................................................................ 15 
5. TREATMENT OF SUBJECTS ............................................................................................ 16 
5.1 Investigational product/treatment .................................................................................. 16 
5.2 Use of co-intervention (if applicable) ............................................................................ 18 
5.3 Escape medication (if applicable) ................................................................................. 18 
6. INVESTIGATIONAL PRODUCT ........................................................................................ 20 
6.1 Name and description of investigational  product(s) ..................................................... 20 
6.2 
Summary of findings from non-clinical studies ......................................................... 20 
6.3 
Summary of findings from clinical studies ................................................................ 20 
6.4 
Summary of known and potential risks and benefits ................................................ 20 
6.5 
Description and justification of route of administration and dosage ......................... 20 
6.6 
Dosages, dosage modifications and method of administration ................................ 20 
6.7 
Preparation and labelling of Investigational Medicinal Product ................................ 20 
6.8 
Drug accountability .................................................................................................. 20 
7. NON-INVESTIGATIONAL PRODUCT ............................................................................... 21 
7.1 Name and description of non-investigational product(s) ............................................... 22 
7.2 Summary of findings from non-clinical studies ............................................................. 22 
7.3 Summary of findings from clinical studies ..................................................................... 22 
7.4 Summary of known and potential risks and benefits .................................................... 22 
7.5 Description and justification of route of administration and dosage .............................. 22 
7.6 
Dosages, dosage modifications and method of administration ................................ 22 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ........................ 22 
7.8 
Drug accountability .................................................................................................. 22 
8. METHODS ......................................................................................................................... 24 
8.1  Study parameters/endpoints ........................................................................................ 24 
8.1.1 Main study parameter/endpoint .............................................................................. 24 
8.1.2 Secondary study parameters/endpoints (if applicable) .......................................... 24 
8.1.3 Other study parameters (if applicable) ................................................................... 25 
8.2 
Randomisation, blinding and treatment allocation ................................................... 25 
8.3 
Study procedures ..................................................................................................... 25 
8.4 
Withdrawal of individual subjects ............................................................................. 25 
8.4.1 Specific criteria for withdrawal (if applicable) ......................................................... 25 
8.5 
Replacement of individual subjects after withdrawal ............................................... 26 
8.6 
Follow-up of subjects withdrawn from treatment ...................................................... 26 

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8.7 
Premature termination of the study .......................................................................... 26 
9. SAFETY REPORTING ....................................................................................................... 27 
9.1 
Temporary halt for reasons of subject safety ........................................................... 27 
9.2 
AEs, SAEs and SUSARs ......................................................................................... 27 
9.2.1 Adverse events (AEs) ............................................................................................ 27 
9.2.2 Serious adverse events (SAEs) ............................................................................. 27 
9.2.3 Suspected unexpected serious adverse reactions (SUSARs) ............................... 28 
9.3 Annual safety report ...................................................................................................... 28 
9.4 
Follow-up of adverse events .................................................................................... 28 
9.5 
[Data Safety Monitoring Board (DSMB) / Safety Committee] .................................. 28 
10. STATISTICAL ANALYSIS……………………………………………………………………..30 
10.1 Primary study parameter(s) ........................................................................................ 29 
10.2 Secondary study parameter(s) ................................................................................... 29 
10.3 Other study parameters .............................................................................................. 29 
10.4 Interim analysis (if applicable) .................................................................................... 29 
11. 
ETHICAL CONSIDERATIONS .................................................................................... 30 
11.1  Regulation statement ................................................................................................. 30 
11.2 Recruitment and consent ............................................................................................ 30 
11.3 Objection by minors or incapacitated subjects (if applicable) ..................................... 30 
11.4 Benefits and risks assessment, group relatedness ........................................................ 30 
11.5 Compensation for injury .............................................................................................. 30 
11.6 Incentives (if applicable) ............................................................................................. 31 
12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .............................. 32 
12.1 Handling and storage of data and documents ............................................................ 32 
12.2 Monitoring and Quality Assurance .............................................................................. 32 
12.3 Amendments ............................................................................................................... 32 
12.4 Annual progress report ............................................................................................... 32 
12.5 Temporary halt and (prematurely) end of study report ............................................... 33 
12.6 Public disclosure and publication policy ..................................................................... 33 
13.  STRUCTURED RISK ANALYSIS .................................................................................... 34 
13.1 Potential issues of concern ......................................................................................... 34 
13.2 Synthesis .................................................................................................................... 34 
14 REFERENCES .................................................................................................................. 35 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; 
in Dutch: Algemeen Beoordelings- en Registratieformulier (ABR-
formulier) 
AE 
Adverse Event 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
DSMB 
Data Safety Monitoring Board 
EU 
European Union 
EudraCT European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
GDPR 
 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics; in Dutch: officiële 
productinformatie IB1-tekst 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; 
in Dutch: Uitvoeringswet AVG 
WMO 
 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 

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SUMMARY 
 
Rationale: There are only limited surgical treatment options like microneurosurgery or biopsy 
for brain tumors. However, very recently several cases have been presented in the literature 
using a cryoablation technique. Cryoablation offers precise and safe lesion targeting with 
minimal blood loss, is minimally invasive and the ice-cone formation within the tumor can be 
monitored by several imaging techniques. Cryoablation could also potentially be used for 
non-resectable tumors. Furthermore, cryoablation could have a potential immunological 
effect in some malignant brain tumors.  Cryoablation could thus be of high value in the 
minimal invasive treatment of several brain tumors.  
 
Objective: This study will be performed to investigate the safety and feasibility of 
cryoablation during operation in adults in several brain tumors like gliomas, meningiomas, 
and brain metastasis. The primary objective is to assess safety in terms of complications rate 
& morbidity and feasibility, such as operation time, blood loss during intervention and 
practicability. The secondary objective is to analyse the confirmation of reaching 
macroscopic tumor edges using intraoperative ultrasound, progression free survival, and 
overall survival during the entire study period. 
 
Study design: This study is set up as an open label, prospective pilot study (Phase I study). 
The study will include 30 patients with gliomas, meningiomas or brain metastasis at the 
ErasmusMC brain tumor center. In all patients, the tumor will be operated conform standard 
procedure in the operation room 2 (Research OR). Upon exposure of the tumor, a biopsy will 
be taken. Subsequently, one or a number of cryoprobes required for tumor ablation will be 
inserted into the tumor. After proper positioning, the cryoablation will start. The ice formation 
will be monitored continuously, using cone-beam CT and/or intraoperative ultrasound. Prior 
to cryoprobe placement, there will be one scan. During cryoprobe placement, a variable 
number of scans may be necessary. After cryoprobe placement, there will be one scan to 
confirm the position. Additionally, there will be one scan during each freezing cycle (1 or 2 
cycles), and one scan after removing the cryoprobes. The duration of each freezing cycle will 
be a maximum of 10 minutes, followed by active thawing. After resection of the necrotic 
tumor, the patients will receive the standard treatment and follow up. 
 
Study population: Adults with presumed gliomas (1. astrocytoma, IDH mutant 2. 
oligodendroglioma, IDH-mutant and 1p/19q-codeleted 3. glioblastoma, IDH-wildtype), 
meningiomas (WHO gr. 1 and gr 2) , or brain metastasis based on preliminary diagnosis.  

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Intervention (if applicable): See study design. 
 
Main study parameters/endpoints: The primary outcome is to assess the safety and 
feasibility of cryoablation in patients with brain neoplasm. Safety will be expressed in terms of 
severity and frequency of the following complications: postoperative intracranial bleeding, 
wound infection, epilepsy, brain edema, neurological deficit, and aphasia. The feasibility will 
be expressed in terms of operation time (in minutes), blood loss during the intervention (in 
milliliters), and practicability. The primary outcome parameters will be compared to standard 
surgery. 
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: All patients included in this study will receive the same (neo-)adjuvant 
therapy (if necessary) as patients who did not participate in this study. Depending on the size 
of the tumor, cryoablation takes approximately 30 minutes to complete. However, we expect 
that the total operating time will be considerably shorter as the cryoablation effect on the 
tumor should shorten the tumor resection time. Cryoablation is already used for the treatment 
of prostate carcinoma, kidney carcinoma and several  lung cancers often with  better results 
than other surgical treatments. Although only few clinical cases have been published, 
cryoablation seems not to increase complications or mortality rates in brain surgery. 
Patient with brain tumors could benefit significantly from this therapy in terms of operation 
time and blood loss.  Cryoablation could also potentially be used for non-resectable tumors 
or very vascularized tumors. Furthermore, the supposed immunological effect could be very 
interesting in combination with adjuvant therapies in the future. 
 

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1. INTRODUCTION AND RATIONALE 
 
The most common types of brain neoplasm are gliomas, meningioma’s and brain metastasis. 
Less common types are vascular tumors such as hemangioblastoma’s and cavernous 
hemangiomas. Frequent clinical presentation of brain tumors in general are seizure, 
cognitive dysfunction, headache and fatigue (1). The diagnosis and treatment response is 
largely evaluated using MRI. Neuroimaging plays an important role in the decision-making 
process for  therapy (2). Depending on many factors, such as the type, size, location of the 
tumor, symptoms and the general health of the patient the main treatment of brain neoplasm 
is usually surgical resection or biopsy followed by radiotherapy and or chemotherapy if 
necessary (3-5). Surgical resection of brain tumors are usually long operations with relative 
high blood loss and high morbidity as compared to non-neurosurgical tumors. The 
development of new minimally invasive safe techniques are therefore warranted.   
 
Recently, several cases have been published by others using a cryoablation technique for 
the treatment of tumors. Cryoablation uses freezing temperatures to destroy tumor  and is 
already an accepted therapy for a variety of tumors like prostate carcinoma, kidney 
carcinoma and several lung cancers often with better results than other surgical treatments 
(6-13). Cryoablation offers precise and safe lesion targeting, is minimally invasive and the 
ice-cone formation within the tumor can be monitored by several imaging techniques. 
Furthermore, cryoablation could have a potential immunological effect in some malignant 
brain tumors. The sharp delineation between tumor and healthy tissue is a big advantage as 
compared to other ablation technique as RF and laser, which makes the treatment of brain 
tumors in eloquent areas with this technique very attractive for neurosurgeons. In other 
techniques as RF and laser ablation, the neurosurgeon cannot properly visualize the ablation 
during the process.  
 
Due to the development of microsurgery and neuroanesthesiology, the clinical applications of 
early crude cryoablation of brain neoplasm, studied mainly in the 60s and 70s were phased 
out (14-19). Only three recent studies reported the use of cryoablation for gliomas and one 
study, for meningioma’s, brain metastasis, and vascular tumors with good results (16, 17, 19, 
20, 22). Martynov et al. (22) treated 88 patients with supratentorial gliomas considered not 
suitable for aggressive surgical resection, and underwent cryo destruction. The complication 
rate of the authors was 11,3% and the postoperative mortality was 1,1%. The complications 
and survival rate of this study corresponded with surgical resection of gliomas in eloquent 
areas  in their clinic (14, 15). However, the study of Martynov et al. was performed by military 
surgeons with a homemade cryosystem and therefore the result could not be extrapolated. 
Gangi et al. (17) treated 4 patients with recurrent glioblastoma using cryoablation. In this 
study no complications were observed. Cebulla et al. (16) also treated patients with 
glioblastoma using cryoablation. One of the six patients that was treated developed transient 
paresis of elevator eyelid muscle. Both Martynov et al. and Gangi et al. froze the tumor twice, 
a so-called double freeze cycle. In contrast to Gangi et al. and Martynov et al. Cebulla et al. 
had only one freeze cycle. Surgical complications following malignant brain tumor surgery 

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has an occurance of 36.2 events per 1000 cases (24). In conclusion, cryoablation shows a 
comparable complication rate as standard brain tumor surgery. 
 
Cryoablation could thus be of high value in the treatment of brain neoplasm. The recently 
published studies are limited and we therefore propose a safety and feasibility study for the 
use of cryoablation in patients with gliomas, meningiomas, and brain metastasis. 
 
 
 

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2. OBJECTIVES 
 
This study aims to investigate the safety and feasibility of cryoablation in adults with gliomas 
(1. astrocytoma, IDH mutant 2. oligodendroglioma, IDH-mutant and 1p/19q-codeleted 3. 
glioblastoma, IDH-wildtype), meningiomas (WHO gr. 1 and gr 2), and brain metastasis. 
  
Primary Objective:  
The primary objective is to assess safety and feasibility in terms of: 
 
Safety: complications & morbidity 
o  Descriptive: Postoperative intracranial bleeding, wound infection, epilepsy, 
brain edema, neurological deficit, death. 
 
Feasibility: Descriptive operation time, blood loss during intervention and practicability 
 
Technical failure 
 
Secondary Objective(s):  
The secondary objective is to analyze: 
 
The conformation of dead tumor cells by pathologist which have been cryoablated 
during surgery  
 
The confirmation of reaching macroscopic tumor edges using intraoperative –
ultrasound (YES/NO). Pictures in 2 directions of the ultrasound will be made to 
objectify 
 
The progression free survival during the entire study period (3 months) 
 
Overall survival during the entire study period (3 months) 
 
Progression free survival and overall survival of the different tumors will be compared 
to matched historic controls. 
 
Safety and feasibility will be compared to historic matched controls 

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3. STUDY DESIGN 
 
This study is an open label, prospective pilot study (Phase I study). Patients will be screened 
for inclusion and exclusion criteria, asked for consent, and when given, enrolled in the study. 
A total of 30 patients with gliomas, meningiomas, or brain metastasis will be included.  
 
This study will include patients who are scheduled to undergo surgery for the resection of 
their brain tumor. All patients will undergo standard neurosurgery (skin incision, trephination, 
and dura opening) to expose cortex with underlying tumor or the tumor directly a vue. A 
biopsy will be taken for pathological diagnosis. After the establishment of the 
histopathological diagnosis, one or more cryoprobes will be inserted into the target tumor. 
Prior to cryoprobe placement, there will be one cone-beam CT-scan. The cone-beam CT is 
located in the operation room 2 where the operations will take place. The number of 
cryoprobes that are required depends upon the size of tumor. Proper positioning of the 
probes will be confirmed using cone-beam CT. During cryoprobe placement, a variable 
number of scans may be necessary. After proper positioning, one scan is needed to confirm 
the position and then cryoablation will start. The growth of the icecone will be monitored 
using intraoperative ultrasound and/or one cone-beam CT. The duration of each freezing 
cycle will be a maximum of 10 minutes, followed by active thawing. A second freezing phase 
(i.e. double freeze protocol) can be chosen if the surgeon deems it necessary. Additionally, 
there will be one scan after removing the cryoprobes. After removing the probes, the necrotic 
tumor will be resected. Tumor remnants will be further examined pathologically. Thereafter, 
the standard closure of the craniotomy will follow.  Patients will receive the standard after 
treatment if necessary. Follow-up is up to 3 months after the treatment of the last included 
patient. A summary of the total study design is provided in Figure 1. 
 
 
 
 
 
 
 
 
 
 
 

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Figure 1- Overview of the study design 
 
 

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4. STUDY POPULATION 
4.1 Population (base)  
Patients will be recruited in the outpatient clinic. Adults with presumed gliomas (1. 
astrocytoma, IDH mutant 2. oligodendroglioma, IDH-mutant and 1p/19q-codeleted 3. 
glioblastoma, IDH-wildtype), meningiomas, or brain metastasis based on preoperative MRI or 
CT diagnosis will be asked for consent, and when given, enrolled in the study by the treating 
physician. 
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
1. Age >18 years 
2. Tumor suspected as glioma (1. Astrocytoma, IDH mutant 2. Oligodendroglioma, 
IDH-mutant and 1p/19q-codeleted 3. Glioblastoma, IDH-wildtype), meningioma 
(WHO gr. 1 and gr 2), or brain metastasis based on preliminary diagnosis for 
which the patient will undergo surgery 
3. Supratentorial or infratentorial localization 
4. Safe trajectory/trajectories possible for ablation of at least 70% of the tumor, 
avoiding eloquent structures 
5. Karnofsky performance scale 70 or more 
6. Sufficient knowledge of the Dutch language to understand the study documents (in 
the judgement of the attending physician or researcher) 
7. Written Informed consent 
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
1. <18 years or >80 years 
2. Tumor diameter bigger than 10 cm 
3. Unsafe trajectory (eloquent structures could be damaged) 
4. Pregnancy 
5. Contra-indication for general anesthesia 
 
4.4 Sample size calculation 
The total of 30 patients is based on the following: only very few clinical studies have been 
published on cryoablation of brain tumors. The study is therefore also explorative. The two 
most recent studies of gliomas included six and four patients respectively. (16, 17) The 
studies of meningiomas and brain metastasis had between 6 to 11 patients each. (18-20) 
Furthermore, there is little published guidance on sample sizes of safety and feasibility trials. 
According to Billingham et al. feasibility studies have a range of 10 to 300 patients per 
arm.(21) Based on other studies about cryoablation in neurosurgery and the audit of 
Billingham et al. we chose a sample size of 30 patients. We expect to include 30 patients 
within 12 months’ time. 

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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
Treatment 
On the evening before surgery 1.5–2.0 mg Lorazepam is administered for anxiolysis. 
60 min. before anaesthesia induction the patient receives 1g Paracetamol p.o. and 7.5-15 
mg midazolam p.o. if requested for sedation. 1g Cefazoline is given iv. for antibiotic 
prophylaxis before anaesthesia induction. 
General anaesthesia is induced intravenously with fentanyl 0.25-0.5 mg, Propopfol 100-200 
mg and cis-atracurium 10-20 mg. After induction of anaesthesia, patient is orotracheally 
intubated and mechanical ventilation is applied. Respiratory rate and tidal volume are 
adjusted to keep the patient normocapnic.  
An arterial line, central venous catheter, and urinary catheter are inserted. Anaesthesia is 
maintained with Propofol (up to 10 mg/kg/h) and Remifentanil (0.5-2 µg/kg/min). Isoflurane 
(up to 1 MAC) and clonidine (1-2 µg/kg) may be added for maintenance, if necessary. The 
fluid management is aiming for normovolemia. 0.9% saline solution and balanced crystalloids 
are used for maintenance, in case of blood loss > 300 ml, HAES 130/0.4 solution will be 
given. 
Temperature management is aiming for normothermia, warm-air blankets and warmed 
infusion lines are used. Arterial blood gas analysis is performed at the beginning of the 
procedure and repeated, if necessary. Electrolytes are controlled and substituted and 
hyperglycemia will be treated with insulin, if necessary.  
The anesthetized patient is positioned on the table. Local infiltration of the scalp is performed 
with 20 ml Lidocaine 1% with Adrenaline 1:200.000 to reduce bleeding. The insertion points 
of the Mayfield Clamp are not infiltrated with local anaesthetics.  
 
After surgical dissection (skin incision, trephination and dura opening) the tumor is exposed. 
Then a biopsy will be taken to confirm the diagnosis. After the establishment of the 
histopathological diagnosis, one or a number of cryoprobes required for tumor ablation will 
be inserted into the tumor. After proper positioning, the cryoablation will start. The ice 
formation will be monitored continuously, using cone-beam CT and/or intraoperative 
ultrasound. Prior to cryoprobe placement, there will be one scan. During cryoprobe 
placement, a variable number of scans may be necessary. After cryoprobe placement, there 
will be one scan to confirm the position. Additionally, there will be one scan during each 
freezing cycle (1 or 2 cycles), and one scan after removing the cryoprobes. The duration of 
each freezing cycle will be a maximum of 10 minutes, followed by active thawing. The final 
step is to resect the necrotic foci as a result of cryoablation guided by STEALTH-
neuronavigation. 
 
At the end of the procedure all anaesthetics are stopped and patient is brought to the Post 
Anaesthesia Care Unit (PACU). Detubation of the patient is performed as early as possible, if 
patient fulfils the detubation criteria (> 36 C body temperature, stable hemodynamics, 
sufficient spontaneous ventilation, adequate response to verbal orders). Postoperative 
analgesia is provided with Paracetamol i.v. or p.o. 1 g up to 4 dd and morphine 7.5 mg s.c. 
up to 4 dd, if necessary. At the PACU the patient is hemodynamically and neurologically 
monitored for 24 hours.  
 
 

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Post-intervention treatment: 
Patients will receive the standard treatment as stated in the treatment protocols. There is no 
objection to radiation or chemotherapy after cryoablation 
 
Products 
 
Cryoablation device:  
For this study, we will use the ICEfx Cryoablation System from the manufacturer Boston 
Scientific. 
 
The ICEfx is the next generation of ablation systems with a compact design. The interface of 
the ICEfx Cryoablation System offers predictable and reliable ablation performance. This 
ablation system is intended for cryoablative destruction of tumor tissue during minimally 
invasive procedures. The ICEfx Cryoablation System is designed to destroy tumor tissue by 
the application of extremely cold temperatures. The system is able to reach temperatures of -
40 °C using argon gas and can thaw the ice formation using helium gas.  
This system is indicated to destroy prostate and kidney tissue, liver metastasis, tumors, and 
skin lesions. According to the manufacturer, the ICEfx Cryoablation device has the following 
indications: 
 
Urology Ablation of prostate tissue in cases of prostate cancer and Benign 
Prostate Hyperplasia (BPH) 
 
Oncology Ablation of cancerous or malignant tissue and benign tumors, and 
palliative intervention 
 
Dermatology Ablation or freezing of skin cancers and other cutaneous 
disorders, Destruction of warts or lesions, angiomas, sebaceous hyperplasia, 
basal cell tumors of the eyelid or canthus area, ulcerated basal cell tumors, 
dermatofibromas, small hemangiomas, mucocele cysts, multiple warts, plantar 
warts, actinic and seborrheic keratosis, cavernous hemangiomas, peri-anal 
condylomata, and palliation of tumors of the skin 
 
Gynecology Ablation of malignant neoplasia or benign dysplasia of the female 
genitalia 
 
General surgery Palliation of tumors of the rectum, anal fissures, pilonidal 
cysts, and recurrent cancerous lesions, ablation of breast fibroadenomas 
 
ENT Palliation of tumors of the oral cavity and ablation of leukoplakia of the 
mouth 
 
Thoracic surgery (with the exception of cardiac tissue) 
 
Proctology Ablation of benign or malignant growths of the anus or rectum 
No specific contraindications are known for the use of ICEfx Cryoablation System. Similar 
systems have already been used in other (European) clinical studies and approved by the 
hospital ethics committees (16-20, 22, 23). Potential side effects that may be associated with 
cryoablation use can be organ-specific or general. 
 
ICEfx uses five different kinds of needles; IceFORCE 2.1, IcePearl 2.1, IceRod 1.5, IceSeed 
1.5, and IceSphere 1.5. Iceball shapes (ellipsoidal or spherical) and sizes are specific to the 
needle brand (Figure 2). There are also needles with different angles available. 
 

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The ice formation around the cryoprobes occur at different temperatures. The first layer 
around the needle is, with -40 °C the coldest, followed by the second layer with -20 °C. 
These two layers are mainly responsible for tumor tissue destruction. The outer layer is 0 °C. 
 
The cryoprobes of this system are supplied sterilized and are not reusable. 
 
 
Ultrasound: 
We will use the BK Medical Ultrasound or Verasonics Vantage-256 functional ultrasound to 
position the cryoprobes and to monitor the formation of the Iceball.  
 
Computer tomography: 
Cone-beam Computer Tomography in operation room 2.  
 
5.2 Use of co-intervention (if applicable) 
Not applicable. 
 
5.3 Escape medication (if applicable) 
Not applicable. 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Figure 2- Iceball shapes and sizes: 
 
 
 
 
 

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6. INVESTIGATIONAL PRODUCT  
6.1 Name and description of investigational  product(s) 
For this study, we will use the ICEfx Cryoablation System from the manufacturer Boston 
Scientific. For more description see 5.1 
 
6.2 Summary of findings from non-clinical studies 
A total of 8 experimental studies used one of the investigational products or other similar 
products for ablation of tumors in glia cell model mice and rats. See figure 3 for a global 
overview of these 8 studies (25-32). 
In summary:  
- 
Cryoablation increased the cellular immunity by activation of dendritic cells 
- 
Cryoablation stimulate the production of specific antitumor antibodies and increase 
the activation of the immune system which may provoke an anti-tumoral response 
 
6.3 Summary of findings from clinical studies 
There are 7 clinical studies were similar products as ICEfx have been used to treat brain 
neoplasm. See figure 4 for a global overview of these 7 studies (16-20, 22, 23).  
 
6.4 Summary of known and potential risks and benefits 
Although only few clinical cases have been published, cryoablation seems not to increase 
complications or mortality rates in brain surgery. 
 
6.5 Description and justification of route of administration and dosage 
Not applicable. 
 
6.6 Dosages, dosage modifications and method of administration 
Not applicable. 
 
6.7 Preparation and labelling of Investigational Medicinal Product 
The cryoprobes of this system are supplied sterilized and are not reusable. 
 
6.8 Drug accountability 
Not applicable. 
<Please describe the procedures for the shipment, receipt, disposition, return and 
destruction of the investigational medicinal products.> 
 
 
 
 

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Figure 3- Global overview experimental studies 
 
 
Figure 4- Global overview clinical studies 

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7. NON-INVESTIGATIONAL PRODUCT 
<This chapter is applicable for any other product that is used in the study, like challenge 
agents or products used to assess end-points in the trial. This can be a medicinal product or 
a food product or a chemical compound or stable isotope or other product. 
This chapter does not include co-medication or escape medication, these are already 
mentioned in chapter 5  
For products to be used as in usual clinical practice the information can be limited to the 
chapters 7.1, 7.6 and 7.7 > 
  
7.1 Name and description of non-investigational product(s) 
7.2 Summary of findings from non-clinical studies 
Not applicable. 
<One may refer to the Investigator’s Brochure (IB), Investigational Medicinal Product 
Dossier (IMPD), Summary of Product Characteristics (SPC) or a similar document (if 
applicable), by mentioning the relevant pages in that document. Be sure that the 
information is up to date and references to peer reviewed papers in (biomedical/scientific) 
journals should be given where appropriate.> 
 
7.3 Summary of findings from clinical studies 
Not applicable. 
<See explanatory text of chapter 7.2, including remark> 
7.4 Summary of known and potential risks and benefits 
Not applicable. 
<See explanatory text of chapter 7.2, including remark> 
7.5 Description and justification of route of administration and dosage 
Not applicable. 
 
7.6 Dosages, dosage modifications and method of administration 
Not applicable. 
 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
Not applicable. 
 
7.8 Drug accountability 
Not applicable. 

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<Please describe the procedures for the shipment, receipt, disposition, return and 
destruction of the non-investigational medicinal products.> 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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8. METHODS 
8.1 Study parameters/endpoints 
The main objective is to assess the safety and feasibility of cryoablation use in patients with 
brain neoplasm. 
8.1.1 Main study parameter/endpoint 
Safety: complications & morbidity during the entire study period as decided by the treating 
physician. Severity and frequency of the following complications:  
 
Postoperative intracranial bleeding 
 
Wound infection 
 
Epilepsy 
 
Brain edema 
 
Neurological deficit (paresis/plegia) 
 
Aphasia 
 
Death 
 
Feasibility:  
 
Operation time (in minutes) 
 
Blood loss during intervention (in milliliters)  
 
Practicability (questionnaire)  
 
8.1.2 Secondary study parameters/endpoints (if applicable) 
 
The confirmation of reaching macroscopic tumor edges using intraoperative 
ultrasound (YES/NO). Pictures in 2 directions of the ultrasound will be made to 
objectify 
 
 
The conformation of dead tumor cells by pathologist which have been cryoablated 
during surgery  
 
 
Progression free survival at the end of the study, estimated at 3 months after surgery. 
The progression free survival is defined as time from diagnosis to disease 
progression (moment of progression as decided by a multidisciplinary team or by 
treating physician) or death, whichever comes first. 
 
 
Overall survival from intervention to end of study (3 months0.  
 
Progression free survival and overall survival of the different tumors will be compared 
to matched historic controls. 
 
Safety and feasibility will be compared to historic matched controls. Outcome will be 
compared using percentages after statistical analysis in the whole group. 

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8.1.3 Other study parameters (if applicable) 
 
Not applicable 
  
8.2 Randomisation, blinding and treatment allocation 
Only patients who comply to inclusion and exclusion criteria are included after informed 
consent. There is no randomization. 
 
8.3 Study procedures 
Prior to operation: 
New patients will be screened for inclusion and exclusion criteria, asked for consent, and 
when given, enrolled in the study. The patient will enroll in the study after the patient has 
signed the informed consent.  
 
Intervention: 
After the standard craniotomy, durotomy, and exposing the tumor as part of a surgical 
procedure, a biopsy will be taken to confirm the diagnosis. Under the guidance of ultrasound 
and/or cone-beam CT, a cryoprobe will be inserted into the tumor tissue. Multiple probes 
may be inserted if necessery. The number of cryprobes depends on the tumor size. After 
positioning the probes, cryoablation system will be set and ice formation will start. The ice 
formation will be monitored continuously. Prior to cryoprobe placement, there will be one 
scan. During cryoprobe placement, a variable number of scans may be necessary. After 
cryoprobe placement, there will be one scan to confirm the position. Additionally, there will be 
one scan during each freezing cycle (1 or 2 cycles), and one scan after removing the 
cryoprobes. The freeze cycle will take a maximum of 10 min followed by active thawing using 
the thawing mode of the cryoablation system. The central temperature of the iceball will be    
-40 °C using argon gas from the ICEfx Cryoablation system. After the freeze cycle, the 
necrotic tumor tissue will be resected.  A summary of the total study procedure is provided in 
Figure 1. Lastly, the dura and the skull will be closed and the patient will receive standard 
postoperative care.  
 
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. 
 
8.4.1 Specific criteria for withdrawal (if applicable) 
Not Applicable. 
 

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8.5 Replacement of individual subjects after withdrawal 
Included patients who underwent cryoablation will not be replaced. When a patient is 
withdrawn by the investigator or withdrawn him/herself before intervention, this patient will be 
replaced if a next eligible patient is available. 
 
8.6 Follow-up of subjects withdrawn from treatment 
There will be no follow up of patients withdrawn from the study. Patients will be treated for 
his/her medical condition outside the study. 
 
8.7 Premature termination of the study 
The study will be terminated: 
 
In case of a very slow inclusion rate (<1 patients per month) 
 
In case of serious adverse events during the intervention  
 
In case of SAEs exceed 50% after every fifth patient is treated 
 
At the request of the sponsor 
 
 

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if 
there is sufficient ground that continuation of the study will jeopardise subject health or 
safety.  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 investigator will take care that all subjects are 
kept informed.  
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 or not considered related to [the investigational product / trial 
procedure/ the experimental intervention]. All adverse events reported spontaneously by 
the subject or observed by the investigator or his staff 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 hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
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. 
A safety monitoring review will be performed after 5/10/15/20/25 patients have been 
treated and followed for one month after the procedure. The higher boundary for the SAEs 
rate is 50% of patients having experienced at least one SAE related to the cryoablation 
treatment procedure. 
 
 
The investigator will report all SAEs to the sponsor without undue delay after obtaining 
 
knowledge of the events. 
 

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The sponsor 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. All adverse 
events will also be reported to Boston Scientific via E-mail 
9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Not applicable. 
9.3 Annual safety report 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC, competent authority, 
and competent authorities of the concerned Member States. 
This safety report consists of: 
 a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
 a report concerning the safety of the subjects, consisting of a complete safety analysis 
and an evaluation of the balance between the efficacy and the harmfulness of the 
medicine under investigation. 
9.4 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.  
 
9.5 [Data Safety Monitoring Board (DSMB) / Safety Committee] 
Not Applicable.  
 
 
 
 
 
 
 
 
 

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10. STATISTICAL ANALYSIS 
We will perform a chi-square test of difference to test whether cryoablation-related 
complications (postoperative intracranial bleeding, wound infection, epilepsy, brain edema, 
neurological deficit, and aphasia) and blood loss during the intervention are related to 
historical matched controls. Normally distributed variables will be summarized using means 
and standard deviations and categorical variables will be summarized using percentages. 
For the primary and the secondary study outcomes, only the available data will be analyzed 
(no imputation of missing data). Only a p-value of <0.05 will be considered to be statistically 
significant. 
 
Statistical analysis will be performed in all 30 patients together to prevent the statistical 
power from getting low. 
 
10.1 Primary study parameter(s) 
Primary study parameters are safety and feasibility. Safety will be expressed in terms of 
severity and frequency of the following complications: postoperative intracranial bleeding, 
wound infection, epilepsy, brain edema, neurological deficit, and aphasia. All data will be 
taken from the patients’ file. The feasibility will be expressed in terms of operation time (in 
minutes), blood loss during the intervention (in milliliters), and practicability. Operation time 
and blood loss will also be taken from patient’s file. For practicality, the neurosurgeons 
performing the cryoablation will be asked fill in a questionnaire, answering questions 
regarding their experiences with cryoablation. The main study parameters will be analysed at 
the end of the study and it will be based on the intention to treat principle. 
 
10.2 Secondary study parameter(s)  
The three secondary study parameters are the confirmation of reaching macroscopic tumor 
edges using intraoperative ultrasound, progression free survival, and the overall survival at 
the end of the study. Progression free survival and overall survival will be analysed by the 
Kaplan Meyer method. 
 
10.3 Other study parameters 
Not applicable. 
 
10.4 Interim analysis (if applicable) 
No interim analysis will be carried out. The researchers do not expect serious morbidity or 
mortality based on literature, and therefore will also not include a stopping rule. 

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11. ETHICAL CONSIDERATIONS 
11.1  Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki and in 
accordance with the Medical Research Involving Human Subjects Act (WMO) and other 
guidelines, regulations and Acts. The project leader is responsible for the proper conduct of 
the study. 
 
11.2 Recruitment and consent 
The attending neurosurgeons will inform patients that may qualify for cryoablation on the 
opportunity to participate in this study. The patient will receive a patient information letter, 
general practitioner letter, and informed consent letter. Patients will have 4 days  to think 
about participating and if necessary more in consultation with the physician or researchers. 
 
11.3 Objection by minors or incapacitated subjects (if applicable) 
Not applicable. 
 
11.4 Benefits and risks assessment, group relatedness 
Cryoablation is used in several medical specialties, often with better results than 
conservative therapies. Cryoablation offers precise and safe lesion targeting, is minimally 
invasive and the ice-cone formation within the tumor can be monitored by several imaging 
techniques.  Cryoablation could also potentially be used for non-resectable tumors. 
Furthermore, cryoablation could have a potential immunological effect in some malignant 
brain tumors. Although very few clinical studies have been published, cryoablation seems not 
to increase complications or mortality rates for brain surgery. Cryoablation could thus be of 
high value in the minimal invasive treatment of several brain tumors.  
Regarding the use of cone-beam CT: by taking additional scans, the placement of 
cryoprobes can be even more precise, increasing the possibility of achieving a larger ablation 
zone. Cone-beam CT is also more suitable for deeper-seated tumors compared to 
ultrasound, making it more feasible in the future. Additionally, cryoablation is often performed 
with the assistance of cone-beam CT in other pathologies. Considering the age (often 60+) 
and limited life expectancy of a significant portion of the patients (including glioma patients 
and patients with brain metastases), it can be assumed that the radiation burden is 
acceptable. 
 
11.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the 
WMO. 
 

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The sponsor (also) has an insurance which is in accordance with the legal requirements in 
the Netherlands (Article 7 WMO). This insurance provides cover for damage to research 
subjects through injury or death caused by the study. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
11.6 Incentives (if applicable) 
Not applicable. 

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
The handling of personal data will comply with the EU General Data Protection Regulation 
and the Dutch Act on Implementation of the General Data Protection Regulation. (in Dutch: 
Uitvoeringswet AVG, UAVG).. Data of each patient will be coded by a unique patient study 
number at enrolment. All data will be collected, stored and locked up in a cabinet by the 
investigational team. Only the investigators will have access to all patient information.  
 
The data will be transferred to a central database by the investigators, which will be 
protected by a password only known to the principal investigator. The backup procedure will 
be followed weakly. 
 
12.2 Monitoring and Quality Assurance  
Monitoring will be done according to monitoring plan B, “Onderzoek met een matig risico”. 
 
12.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the accredited 
METC has been given. All amendments will be notified to the METC that gave a favourable 
opinion.  
 
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 notified to the METC and to the competent authority. 
 
Non-substantial amendments will not be notified to the accredited METC and the competent 
authority, but will be recorded and filed by the sponsor.  
 
12.4 Annual progress report 
The sponsor/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, 

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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 investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as 4 weeks after the intervention of the 
last patient.  
 
The sponsor 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 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.  
 
12.6 Public disclosure and publication policy 
This clinical study will be registered in the Dutch Trail Register before the first patients are 
recruited. The results of the study will be published in the international literature and made 
available to patients who require results. 

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13.  STRUCTURED RISK ANALYSIS  
 
13.1 Potential issues of concern 
Not applicable. 
a. Level of knowledge about mechanism of action 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
e. Analysis of potential effect 
f. Pharmacokinetic considerations 
g. Study population 
h. Interaction with other products 
i. Predictability of effect 
j. Can effects be managed? 
 
 
13.2 Synthesis 
The cryoablation devices that we will use will be used as intended to use. At the start of the 
study, the attending neurosurgeons who will perform the operation will be well trained by the 
manufacturer. All patients included will be operated on as standard treatment followed by 
adjuvant therapy. Exclusively the freezing cycles preceding tumor removal and the 
supplementary CT scans will be conducted for the included patients. The application of cone-
beam CT is proportionate to the condition, age, and average prognosis of the included 
patients. The suitability of cone-beam CT for deeper structures justifies its utilization, as this 
enhances its feasibility for potential future treatments of these conditions and facilitates future 
research endeavors. The complications and mortality of cryoablation are similar to ordinary 
resection. 
 
 
 
 
 
 
 
 
 
 
 

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