High-Intensity Focused Ultrasound (HIFU), a Novel Method for Treatment of Cutaneous Neurofibromas in Neurofibromatosis Type 1: Safety and Efficacy
Phase: NA
Conditions: Cutaneous Neurofibroma
Sponsor: Joergen Serup

 
 
 
 
Clinical investigation plan (CIP) 
 
High-Intensity Focused Ultrasound (HIFU), a novel method for 
treatment of Cutaneous Neurofibromas in Neurofibromatosis 
Type 1: Safety and efficacy 
 
 
 
 
Role 
Name 
Date 
Signature 
 
Coordinating 
investigator 
 
Chief Physician Katrine Elisabeth Karmisholt   
 
 
Principal clinical 
investigator  
Denmark 
 
Sponsor 
 
Professor Jørgen Serup 
 
 
 
Principal clinical 
investigator 
Sweden  
 
Professor Sirkku Peltonen 
 
 
 
 
 
Dokument Ref 
 
cNF-HIFU2101 
CIV-ID 
 
CIV-21-09-037759 
Revision 
 
Ver 2 
Date of version 
 
October 15th  2021 
 
 

 
 
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Table of Content 
 
Preface ............................................................................................................................................ 4 
List of abbreviations and acronyms ................................................................................................. 4 
1 
Identification of CIP .................................................................................................................. 5 
1.1 
Summary of the clinical investigation ............................................................................... 7 
2 
Identification and description of the investigational device ........................................................ 9 
2.1 
System ONE-M ................................................................................................................ 9 
3 
Justification of the design of the clinical investigation ............................................................. 12 
4 
Benefits and risks of the investigational device, clinical procedure, and clinical investigation . 13 
5 
Objectives and hypothesis of the clinical investigation ............................................................ 14 
5.1 
Primary endpoint - Tolerability ........................................................................................ 15 
5.2 
Secondary endpoints – Efficacy, safety and feasibility ................................................... 15 
5.3 
Exploratory endpoints – Diagnosis and measurement methods ..................................... 15 
5.3.1 
Ultrasound imaging assessment and characterization of treated CNF (Denmark) ...... 16 
5.3.2 
Biopsy of Cutaneous Neurofibroma (Sweden) ............................................................ 16 
6 
Design of the clinical investigation .......................................................................................... 16 
6.1 
General .......................................................................................................................... 16 
6.2 
Selection and grouping of tumors ................................................................................... 17 
6.3 
Characterization of treatment response ......................................................................... 17 
6.3.1 
Denmark - High-frequency ultrasound cross-sectional ultrasound imaging................. 17 
6.3.2 
6.3.2 Sweden – Biopsy .............................................................................................. 19 
6.4 
Investigational device ..................................................................................................... 19 
6.5 
Participants .................................................................................................................... 20 
6.5.1 
Inclusion criteria and selection ................................................................................... 20 
6.5.2 
Exclusion criteria for participant selection (non-inclusion criteria) ............................... 21 
6.5.3 
Criteria and procedures for participant withdrawal or lost to follow up ........................ 21 
6.5.4 
Withdrawal from the Study ......................................................................................... 21 
6.5.5 
Study Termination Criteria .......................................................................................... 21 
6.5.6 
Participant enrollment and handling ........................................................................... 22 
6.6 
Procedures .................................................................................................................... 22 
6.7 
Monitoring plan .............................................................................................................. 25 
7 
Statistical design and analysis ................................................................................................ 26 
8 
Data management .................................................................................................................. 28 
9 
Amendments to the CIP ......................................................................................................... 30 
10 
Deviations from clinical investigation plan .......................................................................... 30 
11 
Device accountability ......................................................................................................... 31 

 
 
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12 
Statements of compliance .................................................................................................. 31 
13 
Informed consent process .................................................................................................. 31 
14 
Adverse events, adverse device effects, and device deficiency. ........................................ 32 
14.1 
Adverse Events .............................................................................................................. 32 
14.2 
Serious Adverse Events ................................................................................................. 32 
14.3 
Device deficiency ........................................................................................................... 32 
14.4 
Reporting to national authorities ..................................................................................... 32 
15 
Vulnerable population ........................................................................................................ 33 
16 
Suspension or premature termination of the clinical investigation ...................................... 33 
17 
Publication policy ............................................................................................................... 34 
18 
Bibliography ....................................................................................................................... 35 
 
 
 

 
 
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Preface 
This document is aimed at giving authorities the rationale, objectives, design and proposed analysis, 
methodology, monitoring, conduct and record-keeping of a clinical investigation for ablative 
treatment of cutaneous neurofibromas (cNF) by a new ultrasound therapeutic methodology 
developed for targeted treatment of skin lesions called high intensity focused ultrasound (HIFU). The 
document is structured to follow the outline template for clinical investigation plans stated in Annex 
A of ISO14155:2020. 
The study is conducted in parallel in two centers, one in Copenhagen, Denmark and one in 
Gothenburg, Sweden; each center having special explorative tasks e.g. lesional control by 
noninvasive imaging using 20 MHz ultrasound imaging assessment (Copenhagen) and by punch 
biopsy (Gothenburg). 
An addition, specific assessment of the ethical aspects of the investigation is included in Appendix 
1. This assessment follows and discusses specific issues required by the national ethical committees 
in the two countries where the centers are located. For Denmark ethical issues are governed and 
approved by “De videnskabsetiske medicinske komiteer (VMK)”, while “Etikprövningsmyndigheten” 
governs ethical approvals in Sweden. 
 
List of abbreviations and acronyms 
NF1 
– 
Neurofibromatosis Type I 
cNF 
– 
Cutaneous Neurofibromas 
  
AE 
–  
Adverse Event  
CIP  
–  
Clinical Investigation Plan (according to ISO14155:2020). 
ClinRo –  
Clinician Reported Outcomes 
CRF  –  
Case Report Form  
DK 
–  
Denmark 
GCP  – 
Good Clinical Practice 
HIFU  –  
High Intensity Focused Ultrasound 
IB  
–  
Investigators Brochure 
MDD  –  
European Council Directive 93/42/EEC of 14 June 1993 concerning medical devices 
MDR –  
Regulation (EU) 2017/745 Regulation of the European Parliament on medical devices 
NFD 
– 
Nominal Focal Depth  
OCT  –  
Optical Coherence Tomography 
PRO  –  
Patient Reported Outcome 
QoL 
–  
Quality of Life 
SAE 
–  
Serious Adverse Event  
SE 
–  
Sweden 
US 
–  
Ultrasound Scanning  
 
 
 

 
 
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1 Identification of CIP  
Protocol ID  
cNF-HIFU2101. CIV-21-09-037759  
Study type 
Multicenter (two-center) open-label study in humans (hospital outpatients) 
Study device 
TOOsonix System ONE-M 
A 20 MHz focused ultrasound device for controlled ablative treatment of cNF 
Coordinating Clinical Investigator 
 
 
Katrine Elisabeth Karmisholt 
Chief Physician  
Ass. Professor, PhD, specialist in dermatology 
DK MD registration ID: 00VL2 
Department of dermatology, Bispebjerg University Hospital 
Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark 
Email: katrine.elisabeth.karmisholt@regionh.dk 
 
Principal investigator 
Denmark 
 
Jørgen Serup 
Chief Physician 
Professor, dr. med., specialist in dermatology 
DK MD registration ID: 0094L 
Department of dermatology, Bispebjerg University Hospital,  
Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark 
Email: joergen.vedelskov.serup@regionh.dk 
Phone: +45-21424888 
 
Principal investigator 
Sweden 
 
Sirkku Peltonen 
Professor, MD, PhD, specialist in dermatology 
SE MD registration ID: 7444011 
Department of Dermatology and Venereology, Sahlgrenska University Hospital 
Gröna Stråket 16, 41318 Gothenburg, Sweden 
Email: sirkku.peltonen@gu.se 
Phone: +46-766186260 
 
Investigator Sweden 
 
Despoina Kantere 
Chief Physician 
PhD, Specialist in Dermatology 
Department of Dermatology and Venereology, Sahlgrenska University Hospital  
Gröna Stråket 16, 41318 Gothenburg, Sweden 
 
GCP support  
Denmark 
(pending contract) 
 
GCP-enheden ved Københavns Universitetshospital 
Frederiksberg Hospital 
 
Nordre Fasanvej 57, Skadestuevej 1, parterre, 2000 Frederiksberg 
E-mail:gcp-enheden.bispebjerg-frederiksberg-hospitaler@regionh.dk 
Tlf: 38 63 56 20 
 
GCP support 
Sweden 
(pending contract) 
 
Gothia Forum for Clinical Studies  
Research Support Office  
Sahlgrenska University Hospital 
Guldhedsgatan 10C 
 413 46 Göteborg  
Email: gothia.forum@vgregion.se 
Tel: +46 31 342 96 70 
 
 

 
 
 
 
Table 1. Planned activities in the clinical investigation. The investigation consists of a single treatment session (Visit 2) with 3 subsequent follow-up 
evaluations. The first data analysis and report on primary end-points will be done as an interim assessment after the 3-month visit has been completed by all 
participants (Visit 4). Primary and secondary end-points are concluded in a final report after all participants have completed the 9-month visit (Visit 5). 
Visit number 
Event Name 
1 
Pre-study 
2 
Day 0 
3 
7 ± 3 days 
(1 week) 
4 
12 ± 2 weeks 
(3 months) 
5 
24 ± 2 weeks 
(6 months) 
6 
39 ± 3 weeks 
(9 months) 
 
Consent, Screening 
and Enrollment 
Pre-treatment 
documentation, 
treatment, and post-
treatment 
assessments 
Evaluation of wound 
generation  
Evaluation of healing 
and surveillance for 
any new lesions 
Evaluation of healing 
and surveillance for 
any new lesions 
Evaluation of healing 
and surveillance for 
any new lesions 
Clinical evaluation 
X 
X 
X 
X 
X 
X 
Inclusion and registration 
X 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
Photo documentation 2D 
 
X1 
X 
X 
X 
X 
Photo documentation 3D 
 
X1 
X 
X 
X 
X 
Ultrasound Imaging Assessment (Bispebjerg Hospital) 
 
X1 
X 
X 
X 
X 
HIFU treatment 
 
X2 
 
 
 
 
Participant questions and scoring  
 
X1 
X 
X 
X 
X 
Medical team questions and scoring 
 
X1 
X 
X 
X 
X 
Adverse Event Reporting  
 
X1 
X 
X 
X 
X 
Biopsy of 1-3 treated cNF3 (Sahlgrenska Sjukhuset) 
 
 
X 
X 
 
 
Biopsy of 1 untreated cNF (Sahlgrenska Sjukhuset) 
 
 
X 
 
 
 
1 = Data collection should occur immediately before treatment and again within one hour after treatment  
2 = A minimum of 5 cNF 2-5 mm in size are required for enrollment. A minimum of 3 and a maxumum of 9 cNF will be treated and assessed across visits 2-6 
3 = The minimum number of cNF to be biopsied is 3 (two treated and one control). The maximum number of cNF to be biopsied is 7. The biopsies will occur for cNF that are between 2-5 mm in 
size at visits 3 and 4. There will be one untreated cNF biopsied at visit 3. A minimum of one treated cNF will be biopsied at both visit 3 and visit 4.  
 

 
 
 
 
1.1 Summary of the clinical investigation  
The overall objective of this study is to evaluate the safety, tolerability, and efficacy of cutaneous 
neurofibromas (cNF) removal in adults with Neurofibromatosis Type 1 (NF1) using high-intensity 
focused ultrasound (HIFU). 
With an incidence of 1/2500 to 1/3500, Neurofibromatosis Type I (NF1) is among the most common 
single-gene inherited conditions worldwide (1, 2). The genetic condition predisposes to multiple 
forms of histologically benign and malignant neoplasms. The most common tumor across all people 
with NF1 is cutaneous neurofibroma (cNF), impacting up to 99% of adults (3-7). These tumors can 
number in the hundreds to thousands on a given individual, most often appear between late school 
age and in the second decade, and increase in number and size across the lifespan (3-5, 8-12). 
In many patients, cNF are socially debilitating, painful and impede activities of daily living (4, 5, 10-
14). Multiple different appearances of cNF exist as reported from patients and clinicians. These can 
range from barely visible flat nodules with subtle discoloration, to large and pedunculated masses. 
One proposed classification system to describe the clinical appearance of cNF assigns one of five 
subcategories: nascent, flat, sessile, globular, or pedunculated (15). Nascent lesions (very small 
dispersed tumors) and peduncular neurofibromas (protruding tumors with a narrow base) are not 
subject to study as they are not logic candidates for HIFU treatment at this early stage. The typical 
appearance of the cNF-categories relevant for treatment in this study is illustrated in Fig. 1. 
Despite the remarkable prevalence of cNFs in people with NF1 and their documented influence on 
quality of life (QoL), current treatment is limited to surgical removal or the use of various devices that 
cause tissue destruction showing mixed, but largely low-efficacy success, to date (11, 16-18). 
While blade-based surgical removal allows complete removal of the lesion(s), this method is 
impractical to address the many tumors affecting a typical adult with NF1, thus forcing patients to 
select specific tumors to remove. Additional drawbacks of surgical resection include variable 
scarring, and the time required for the procedure and healing. Alternative approaches that enable 
the removal of larger numbers of lesions per session include electrosurgical ablation, laser-based 
treatments, and radiofrequency ablation all reported to have variable outcomes(11, 15-17, 19-22). 
 
 

 
 
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Fig. 1. Three subtypes of cutaneous neurofibromas considered for the current study. 
The investigational device used in this study is a Danish developed system. As the first of its kind in 
the world, it is capable of controlled and targeted thermo-mechanical treatment to small intradermal 
volumes containing neoplastic cells, without inflicting damage to the surrounding tissue (23-26). 
The device is proposed to be used in a non-ablative modality, where treatment does not damage the 
skin’s basement membrane, and the skin surface thereby remain intact. Healing after treatment will 
thus be facilitated by internal cell transport through the vasculature, and risks of post-treatment 
complications or visible scarring will be minimized (23-25, 27). 
The current protocol presents an investigation with an evaluation of the safety and efficacy profile 3 
months after a single 3-5 minute treatment. Subsequent follow-up of secondary endpoints is done 
every third month until the end of the study 9 months after the treatment. The activities of the 
investigation are summarized in Table 1, and a graphic presentation of the patient-flow and actions 
are given in Fig. 2. 
Provided that the expected positive clinical results can be verified, this new method could have 
potential for widespread use as an important replacement or compliment to existing methods for 
treatment of cNF. 

 
 
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Fig. 2. Study flow chart. The study is an initial tolerance-study with the objective to investigate if a new 
therapeutic modality is safe, efficient and feasible, and thereby should be investigated further in larger 
future studies. cNF to be treated can be anywhere on the body with the exception of face, shoulders and 
the breast. 
 
2 Identification and description of the investigational device 
2.1 System ONE-M 
The investigational device, called TOOsonix System ONE-M, is described in detail in the 
investigator’s brochure, which includes details on model, type, software, accessories, the 
manufacturer, and the regulatory status of the device authorized for clinical studies on humans. The 
device is shown in Fig. 3. 
 

 
 
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Fig. 3. TOOsonix System ONE-M; 20 MHz focused ultrasound applied to skin with a selection of 
handpieces (depth of focal point in the skin 0.8, 1,3, 1,8 and 2,3 mm). Treatment is surveyed with a 
camera incorporated in the handpiece. 
 
The operating principle of focused ultrasound has been used for decades for non-invasive treatment 
of lesions in internal organs (28-33). Acoustic energy is generated in a transducer with a concave 
geometry allowing the emitted sound to concentrate in a high-energy focal zone inside the body, 
where a thermal lesion can be positioned in the target of interest chosen to be treated. 
Depending on instrument construction and setting, short pulses can produce a controlled thermal 
insult in the target. This target could be; e.g. a kidney stone or a cancer as exemplified by a breast 
cancer, thyroid cancer or a prostatic carcinoma resulting in thermal necrosis and destruction of the 
target. Focused ultrasound can also be used for treatment of intracranial targets, such as centers 
responsible for essential tremor and Parkinson’s disease (26, 31, 34). The HIFU operating principle, 
which illustrates the application to skin, is shown in Fig. 4. 
The convergent ultrasound beam, on its passage through tissues to reach the focal point, with proper 
settings, will not exert damage to the passage medium of tissue between transducer and target. 
Thus, the necrotizing or ablating effect can be set to match a desired level in the skin without causing 
effects in the adjacent tissue above, below and around. 
Focused ultrasound devices, including the device under study, are designed to produce a focal 
heating in a focal point reaching up to 65 °C. This is a level that is high enough to produce cellular 
death in the focal zone, but low enough not to transmit major caloric energy to the surrounding tissue 
(23, 24).  
 
 

 
 
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Fig. 4. Schematic illustration of the HIFU principle with concentration of the ultrasound energy inside the 
skin in a focal point exposed to a thermal insult causing cell death. 
 
Treatment of internal organs with focused ultrasound has been documented in clinical research, and 
the method is now used around the world in oncology clinics of excellence (26). Treatment of internal 
targets in the body requires deep penetration and therefore need to operate with longer wavelengths 
at lower frequencies (from 500 kHz to 5 MHz), where attenuation of ultrasound is relatively low. For 
the treatment of dermal lesions, much smaller wavelengths at higher frequencies (> 15 - 20 MHz) is 
however needed, since targets, such as skin tumors, have small dimensions and need selective 
treatment with a small and well-controlled focal point both vertically and horizontally (23). 
As the TOOsonix device is the first device operating at 20 MHz, the resolution allows for selective 
treatment of small sized targets in the skin. The device has shown efficacy in other diseases limited 
to the skin in which the goal was to eradicate the neoplasm without damaging the epidermis. There 
is therefore potential that the TOOsonix device can address multiple sizes and forms of cNF without 
the burden of scarring. 
Practical operation of the investigational device is comparable to conventional dermatological lasers, 
which are covered in the formal specialist courses dermatologists must pass. An introductory training 
in focused ultrasound is needed, with practical training performed on some suitable tissue-like 
objects. TOOsonix A/S is offering introductory information, theoretically and practically, at the start 
of study, including instruction in the use of different hand pieces with different penetration depths. 
During operation, a treatment dose of typically 150 milliseconds duration is applied to the skin under 
real-time supervision using the integrated camera showing a magnified image of the skin surface. 
Repeated ultrasound dosing is administered in close proximity to each other and positioned to cover 
the entire skin lesion in a “shoulder-by-shoulder” pattern. At the time of a dose-activation, a circular 
shockwave-effect on the outer skin is displayed, and it can be monitored and adjusted such that an 
applied dose is not too weak and not too strong. 
Depending on the choice of hand piece (focal depth) used on the investigational device, the 
treatment can be selected to be ablative, with transport of dead cells into a superficial wound crust 
through a damaged basement membrane, or non-ablative with internal transport of dead cells 
through the body’s vasculature. 
The device will be applied to cNFs that are intradermal. Visible cNFs commonly vary in diameter 
from 1 mm up to 1 cm in size. cNFs are, irrespective of size, typically covered by a roughly 150 µm 

 
 
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thick epidermis and 200-500 µm thick rim of dermis. Neurofibromas are by histology composed of 
Schwann cells, and are variable connective tissue components in a loose and poorly defined 
structure. 
The treatment in this study is thus aimed at use in the non-ablative modality, where cell death in the 
dermis is created without damaging the basement membrane, subsequently minimizing the risk of 
broad tissue damage to dermal collagen, and thereby reducing unintended scar formation after 
healing. 
To obtain the non-ablative treatment modality, hand pieces with nominal focal depth (NFD) depth of 
1.8 mm or 2.3 mm will likely be the preferred standard in this study, while allowing for discretion of 
the investigator to use any available hand piece deemed appropriate based on the pre-treatment 
assessment of the thickness and invasion level of the tumor in the skin. Two more superficial hand 
pieces with NFD of 0.8 mm and 1.3 mm are available with the standard device accessories from the 
manufacturer.  
The device displays the instrument settings and the number of shots applied during a treatment 
course, thereby relieving the operator of extraneous distractions. Additionally, the device works by 
ultrasound as the sole modality, relieving the patient the undue burden of additional medicines, 
electromagnetic radiation, or other more invasive measures. 
3 Justification of the design of the clinical investigation 
In this study, the TOOsonix System ONE-M is applied to cutaneous neurofibromas (cNF) consistent 
with neurofibromatosis type I (NF1).  
Given the very low number of available dermatologic agents for the treatment of cNF, and the lack 
of data about the specific performance of these instruments and formal clinical assessment, this 
study will systematically assess the TOOsonix device for tolerability, biologic effect and efficacy as 
assessed by patient reported outcome and image assessment before and after treatment. This 
data will help determine if additional study of the TOOsonix device is warranted for cNF in people 
with NF1. 
The investigational device is a Danish developed system aimed specifically at high frequency 
operation (20 MHz). At this high frequency, the device, as the first of its kind, allows controlled and 
targeted treatment of small intradermal volumes, thus with limited damage to the surrounding tissue. 
The investigational device has passed pre-clinical studies in experimental laboratory models and in 
a safety study on Göttingen minipigs as reported in the IB and published in the literature (23, 24). 
The pre-clinical experiments showed the following key results and conclusions: 
• 
The 20 MHz ultrasound output is a well-defined ultrasound cone with a sharp focal point as 
demonstrated in laboratory measurements and in tissue-mimicking phantom gel simulating 
skin. 
• 
The produced thermal lesion is highly reproducible from dose to dose. 
• 
The depth of the focal point can be chosen and positioned as intended depending on the 
selected hand piece. 
• 
The temperature in the focal point reproducibly matches 65 °C. 

 
 
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• 
The extension/severity of the heat effect in a phantom and read as necrotizing effect and 
heat lesion in the pig study was tunable within the range of available instrument settings, and 
reproducible. 
• 
Depending on device setting, non-ablation subclinical effects can be induced, alternatively 
ablation/necrotizing effects, the former being relevant for cNF therapy. 
• 
Post-treatment wound and healing studied in the pig followed over time (12 weeks) depended 
on the applied ultrasound energy. 
• 
Isolated single dosing had much lower, or no, necrotizing effects as compared to multiple 
shoulder-by-shoulder dosing applied to a larger field. 
• 
Histology after three months indicated mild fibrosis in pigs treated with higher ultrasound 
energy, however, clinical scar formation was not visible, or only barely visible. 
• 
Studies indicated the depth of the ultrasound lesion in the dermis are well controlled by way 
of proper selection of instrument settings, and the choice of hand piece. 
• 
Depending on dose, a transient wheal and flare reaction, with redness and edematous 
swelling, occurred within minutes and faded within about 30 min. This reaction is comparable 
to skin reactions from dermatological lasers. The reaction is considered to be caused by 
traumatic histamine release. 
• 
Ultrasound treatment in clinical use may be associated with instant pain and possibly itch. 
• 
Studies indicated no unexpected adverse events, and no systemic safety concerns. 
 
The skin lesions observed in the pig models and human treatments therefore justifies the present 
protocol addressing treatment of human cNF, which are superficial skin lesions of small size, and 
easy to cover with visually guided shoulder-by-shoulder applied ultrasound shots. 
This study is the original and the first systematic study of 20 MHz focused ultrasound applied to NF1 
associated cutaneous neurofibromas. The method has the potential to open a new venue of clinically 
useful tumor mitigation therapy in people with NF1 where there is a major unmet need. 
4 Benefits and risks of the investigational device, clinical 
procedure, and clinical investigation 
The anticipated benefit of the 20 MHz HIFU modality in treatment of benign neurofibromas is to 
develop and confirm a new targeted therapy with a high benefit-risk ratio compared to existing 
surgical methods. 
Cutaneous NFs for treatment will be selected in agreement with patients. Visible skin areas will be 
avoided since scar-formation resulting from HIFU treatment of cNF is not yet documented. 
Specifically, lesions on the face, shoulders and breasts will not be treated as these areas are 
especially vulnerable to development of scaring. 
Participants shall be informed about potential risks and discomforts of the HIFU treatment before the 
treatment is applied. Persons having psychological or neurologic risk of pain are not included in the 
study. Persons with any acute or chronic medical condition, which in the opinion of the investigator, 
could interfere with this study are also not included in the study. 
At the Danish center, the study will include ultrasound imaging assessment before and after HIFU 
treatment. This method is non-invasive and known to be without risks to the patients.  
At the Swedish center, the study includes a minimum of 3 and a maximum 7 small punch biopsies 
taken from control and treated lesions during the study. Topical anesthesia will be used according 

 
 
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to normal routine before biopsy. Biopsies taken on cNFs are part of the routine treatment regime, 
and the overall risk is known to be minor and serious adverse events are very unlikely. 
Focused ultrasound applied as short doses producing 65 °C heat in a cancer is widely documented 
in the literature as having a necrotizing effect on internal cancer tissue followed by clinical healing or 
cancer reduction (28-34). It is expected that the HIFU lesions in skin are equally sensitive to 
ultrasound generated heat. 
The expectation regarding treatment of skin is therefore high, since these tumors are small and 
superficial, and therefore visible and easy to diagnose and target. The treatment effect can be dosed 
precisely under real-time visual supervision from the investigational device. 
Anticipated local adverse effects are immediate wheal and flare reactions at the treatment site, and 
complications of wound healing, including the potential risk of bacterial infection and scarring. The 
former is significantly reduced when treatment is administered in the proposed non-ablative modality, 
where the skin barrier remains intact, and bacterial access thereby remains closed. Risk of scarring 
after healing of insults to the lower half of the dermis is known, but also depends on anatomical site 
and constitutional factors (24). Compared to potential scarring from healing of open surgical wounds 
the visual result is however considered to be very beneficial. 
The treatment carries very low potential risk of systemic adverse effects except for pain on treatment 
due to heating in the treatment zone. Pain has an instant onset, but is very short, since the preferred 
pulse duration is only 150 milliseconds. However, participants shall be instructed about pain of 
treatment before treatment is provided. 
Histamine release in the treated area may cause wheal-and-flare associated with itch on the treated 
site, however, traumatic wheals are short-lived and fade over 20-30 min (24). HIFU is, in comparison 
to light, not expected to interfere with the effect or metabolism of circulating drugs.  
Finally, HIFU is a physical acoustic wave that can be transmitted through coupled media only. Unlike 
in laser treatments, users or patients are therefore not at risk of any unintentional exposure. The 
treatment can thus be conducted in a normal clinic setting without use of eye-protection PPE etc. 
The results of this research may gain further understanding of use of these treatments in providing 
a new tool for treating cNF and potentially helping prevent growth of such lesions. There may or may 
not be direct benefits to participants in this study, however previous trials showed improvement in 
lesion appearance/clearance, as well as high patient satisfaction. 
5 Objectives and hypothesis of the clinical investigation 
The hypothesis of this study is, that small and highly localized acoustic focal zones produced by a 
new HIFU device non-invasively can induce acute cell-death in cutaneous neurofibromas (cNF) 
without disrupting the skin’s basement membrane. The healing of the treated volume will 
subsequently be facilitated by internal cell-transport through the vasculature without creation of open 
wounds. This therapeutic modality will thus give less post-treatment complications and less visible 
scarring compared to surgical procedures that are currently the only standard treatment for removal 
of cNFs in patients carrying the Neurofibromatosis Type 1 genetic condition. 
The objective of the study is therefore to document that 20 MHz focused ultrasound is safe and 
effective in the treatment of NF1 associated cNF with high reduction of the measured volume of each 
treated tumor after 3 months, and with follow up registration of potential regrowth or development of 
new tumors in the treated and untreated regions at 6 and 9 months. 

 
 
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5.1 Primary endpoint - Tolerability 
• 
The grade of any adverse events (AE) requiring medical intervention within three months of 
treatment should be low. Device-based treatment will be considered tolerable if less than 
30% of participants treated have an “Overall AE Score” larger than Grade 2 AE at the 3-
month follow-up (visit 4) as assessed via Common Terminology Criteria for Adverse Events 
(CTCAE), U.S. Department of health and human services, Ver 5.0, Nov 27, 2017. 
 
• 
The rate and nature of spontaneous healing of the treated cNF lesion and any safety related event 
including potential wound formation and wound healing should be equivalent to expectations from 
alternative method(s). Safety evaluation includes rating of treatment associated sequele by end 
of study, e.g. dyspigmentation and scarring and overall investigator rating compared to 
expectations. 
 
5.2 Secondary endpoints – Efficacy, safety and feasibility 
• 
Efficacy: Rate of clearance, e.g. reduction of tumor mass of treated cNFs based on 2D and 
3D photography or ultrasound imaging assessment at 3 and 9 months parallel to assessment 
of the degree of dyspigmentation with hypermelanosis or hypomelanosis of the lesion and 
the perilesional untreated skin. 
• 
Safety: Itch and pain from the treatment measured on a 0-10 point numerical scale. 
• 
Safety: Wheal and flare reaction and inflammatory responses are measured on a graded 
scale. 
• 
Safety: Any objective adverse effect or event, local or systemic, related to the treatment and 
the investigational device is measured (wound and course of wound healing, scar formation, 
instrumental hazards) is categorized immediately after treatment and at all follow-up visits. 
• 
Feasibility: Feasibility is evaluated both quantitatively (time spent on treatment) and 
qualitatively depending on the object considered and cover elements that may compromise 
or favor HIFU relative to other treatments employed for lesions in clinical practice or from the 
literature. Feasibility assessment will not produce a direct comparison with alternative 
methods since the study has no arm with an alternative method. The evaluation comprise 
the following essentials: 
1) Time and resource spent on the treatment session using the device, 
2) Investigators’ assessment of the practice of using the device and the treatment, 
3) Participants’’ assessment of satisfaction with the treatment and the device. 
 
All skin toxicities will be grated according to the classifications in Common Terminology 
Criteria for Adverse Events (CTCAE), U.S. Department of health and human services, Ver 
5.0, Nov 27, 2017. 
 
5.3 Exploratory endpoints – Diagnosis and measurement methods 
A core question to be addressed in this study is the effects of the thermal energy delivered to the 
treated cNF. Specifically, the study will seek to understand if the entire cNF tumor receives equal 
thermal energy by the method, if there are differences in dose relative to depth of the lesion, and 
what the inflammatory response within and around cNF is at one week (in the early stage of reaction) 
and after 3-6 months when coagulation and connective tissue repair are expected to be completed. 

 
 
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The inflammatory infiltrate observed after one week will indicate the severity and extension of the 
HIFU thermal effect on the cNF, and it can be assesses if the entire lesion was given full treatment 
or not. This data will be necessary to select recommended instrument setting and dose interval in 
future efficacy studies. 
Both centers will assess the tumor’s horizontal extension and skin elevation including typing (flat, 
sessile, globular) before and after treatment using 2D and 3D digital images as a part of the 
secondary efficacy endpoint. Additional exploratory end-point for qualitative and quantitative 
evaluations of the above characteristics and response to the method will be based on existing 
expertise and routine at the respective centers. 
 
5.3.1 
Ultrasound imaging assessment and characterization of treated CNF (Denmark) 
Ultrasound imaging assessment will be used to assess the vertical thickness and depth of tumors at 
each time point of the study using a 20 MHz B-Mode ultrasound scanner. The scanner allows non-
invasive measurement of tumor depth, cross-section area and quantitative characterization of e.g. 
various inflammatory responses. Measurements made on the tumors before HIFU, immediately after 
treatment, and at all follow-up visits will be noted directly in the study CRF. The combined 
conclusions regarding the use of ultrasound imaging assessment as a method for assessing the 
safety and efficacy of the HIFU treatment of cNF will be summarized by the center investigator in the 
final study report. 
 
5.3.2 
Biopsy of Cutaneous Neurofibroma (Sweden) 
In Sweden, treated and reference untreated cNF from the same anatomical region will be subjected 
to biopsy and histological evaluation as a means of characterizing the above post-treatment 
characteristics. Small punch biopsies are preferred, but the investigator may decide to perform a 
surgical biopsy, or a shave biopsy, depending on participant’s preference and the aim to possibly 
remove the entire lesion with the biopsy. 
After the first three patients have been treated and have had the 3-month biopsy completed, the 
tissue will be assessed by histopathologists to assess if the pathology is stationary or not. The 
combined conclusions regarding the use of biopsy as a method for assessing the safety and efficacy 
of the HIFU treatment of cNF will be summarized by the center investigator in the final study report. 
 
6 Design of the clinical investigation 
6.1 General 
The study is a two-center, open-label study in humans (hospital outpatients). 
The two centers are both located in EU, and thus follow the same overall regulatory framework for 
clinical research and approvals. 

 
 
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The center located in Denmark, Department of Dermatology, Bispebjerg Hospital Region 
Copenhagen Capital, is a department under the Copenhagen University. The department provides 
advanced dermatological treatment, and is the largest dermatology department in Denmark. 
The center located in Sweden, Sahlgrenska University Hospital is a non-profit University hospital 
owned and managed by Västra Götaland Region. It is a secondary referral center for dermatological 
patients and serves a population of about 750 000 in and around city of Gothenburg. 
6.2 Selection and grouping of tumors 
Tumors to be included in the study will be selected and documented in the treatment session (visit 
2) via collaboration between the participant and the treating physician. According to the willingness 
of the participant there may be 1-3 triplets of tumors representing all three categories (flat, sessile 
and globular) which would make a total of 3-9 cNF tumors treated and 3-7 cNF tumors biopsied per 
participant. The minimum number of cNF to be treated is three. The maximum number of cNF to be 
treated is 9. The minimum number of cNF to be biopsied is 3 and the maximum number of cNF to 
be biopsied is 7 to limit discomfort or inconvenience associated with healing. Ideally, paired sets of 
tumors representing all three categories (flat, sessile and globular) will be included. However, it is 
acknowledged that this may not be feasible to identify all three categories of tumors in pairs in each 
participant. It will be noted in the CRF which cNF is the control/reference and which are treated and 
sampled. The selected tumors will be documented by 2D and 3D photos. 
 
6.3 Characterization of treatment response 
The two centers will use identical clinical criteria of diagnosis of cNF which is a clinical diagnosis 
made in a person with NF1 who has skin neoplasms involving the dermis (see Fig. 1). 
The cNFs to be treated will initially be described relative to anatomical site, tumor size at baseline, 
cNF subtype (Fig. 1), pigment, hair, and associated symptoms and signs at the time of treatment. 
The rationale for selection of tumors will be described (pain, itch, disfigurement). The selected tumors 
will be documented by 2D and 3D photos. 
The centers will furthermore use two different methods to document the base-line state of the 
selected cNFs, and the response and result of the treatment immediately after treatment, and in the 
subsequent follow-up visits. Each method is well established in the respective department routine. 
In addition to the digital images, in Denmark cNF will be measured with 20 MHz pulsed ultrasound 
imaging and in Sweden a selection of cNF tumors will be biopsied (see section 6.2 and 6.3.2). 
6.3.1 
Denmark - High-frequency ultrasound cross-sectional ultrasound imaging. 
In Denmark, measurements of the baseline state of cNFs to be treated, and subsequent response 
to the treatment will be based on 20 MHz pulsed ultrasound imaging. 
20 MHz ultrasound assessment has been used in many fields in dermatology for measurement of 
skin and tumor thickness and for characterization of inflammatory skin diseases since its introduction 
in 1979. Tumors and inflammation create low echogenic vertical profiles in the dermis, which can be 
measured by ultrasound imaging, including an analysis of the image density registered as low 
echogenic pixels (27). 

 
 
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The DermascanC, from Cortex Technology, Hadsund, Denmark will be used. Ultrasound imaging 
assessment with this system creates real-time, cross-sectional images to a depth of up to 4 mm with 
axial resolution about 25 µm and lateral resolution about 70 µm. The ultrasound pulses are of low 
energy and involves no safety concerns related to persons being scanned, and has no therapeutic 
effect confounding the HIFU treatment.  
Scanning is non-invasive and not associated with any pain or discomfort. In this study the manual 
method of recording in vivo distances and thickness will be applied, and not the automatic facility 
having less accuracy (27). An example of an ultrasound imaging of a dermal tumor (not cNF) with 
qualitative measurement of the cross-sectional area is shown in Fig. 5. 
 
 
Fig. 5. Example of measurement of a tumor cross-section using the Cortex DermaScan C device (not 
cNF). Source: https://cortex.dk/scientific-ultrasound-dermascan-c/ 
 
Ultrasound imaging assessment will be performed on all treated and control cNFs included in the 
evaluation at baseline (visit 2), one week after treatment (visit 3), 12 weeks after treatment (visit 4), 24 
weeks/6 months (visit 5) and 39 weeks/9 months (visit 6). This will be done concurrent with 2D and 3D 
photos (Table 1).  
 

 
 
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6.3.2 
6.3.2 Sweden – Biopsy 
In Sweden, patients will undergo biopsy of a minimum of 3 cNFs (1 control/reference, 1 cNF biopsied 
one week after treatment, and 1 cNF at 12 weeks after treatment) and a maximum of 7 (1 control 
and 6 treated cNF) to characterize the baseline and treated response at two time points (1 and 12 
weeks after treatment) (Fig. 6). 
The control biopsy will be taken one week after the treatment (visit 3). Biopsies of the treated tumors 
will be taken one week after treatment (visit 3) and 3 months after treatment (visit 4). The pathologic 
analysis of these tumor samples will illustrate the short-term inflammatory response and mid-term 
healing effects respectively. If the patient is willing to have more tumors treated and biopsied, a 
maximum of 1 control biopsy and up to 6 biopsies from treated tumors per patient will be taken 
throughout the study. The schedule for biopsy characterization is illustrated in Fig. 6.  
Tumors subject to biopsy are lost for clinical follow up, thus, up to nine lesions per patient will 
preferably be included in the study in the Swedish center. cNFs selected for biopsy must be 2-5 mm 
in diameter. Biopsy is preferably taken as a punch biopsy, but the investigator may decide to perform 
a surgical biopsy or a shave biopsy depending on participant’s preference and the aim to possibly 
remove the entire lesion with the biopsy. 
The tissue collected via biopsy will be assessed with standard histology and immunohistochemistry.  
 
Fig 6. Schema for cutaneous neurofibroma biopsies.  
 
6.4 Investigational device 
The investigational device, TOOsonix System ONE-M, was CE-marked as an electrical medical 
device according to MDD as a Class I device in July 2020. The device therefore conforms to the 
relevant safety requirements needed for human treatments in the harmonized IEC standards 
covering medical devices. Type testing was performed by TÜV SÜD, with a range of supporting 
evaluations and tests performed by internal and external parties. 

 
 
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The device is indicated for a range of aesthetic procedures, and has furthermore been used in case-
based treatments, on the doctor’s clinical responsibility, for selected clinical volunteers that could not 
helped by other treatments. 
Recently a GCP clinical Investigation on removal of non-nodular basal cell carcinoma using the 
device was submitted and conditionally approved by the Danish medical and ethics authorities. The 
study is planned to start in the autumn 2021 after all final relevant contracts and have been signed. 
During operation, the HIFU device is not in contact with the participant except for the touch from the 
tip of the hand piece held against the skin surface, with a standard ultrasound gel used as coupling 
medium between skin and probe. There is no electrical contact between device and participant. The 
contact involves no physical insult, cut or injection, and no breakage of the skin barrier. Probe tip 
and cables are validated allergen free and biocompatible. 
No comparator device is included in the study, and no medicinal product is involved. 
6.5 Participants 
Study participants are recruited among the hospital patients of the two centers, invited out of the 
routine offering them different treatment options. Further recruitment may be made by direct referral 
of eligible patients after contact to other relevant hospital departments and centers or patient 
associations in these the respective countries. 
Diagnosis of their cNF is according to standard routine of the hospitals. Confirmation of the 
neurofibromatosis syndrome by biopsy is not used in the routine since the disease is characteristic 
and commonly diagnosed by simple means e.g. history and clinical signs. Patients may have been 
evaluated genetically or not. Participants’ enrolment as hospital patients continues along with their 
participation in the study. 
Because most patients with NF1 and cNF wish many of their cNFs treated, there is no upper limit of 
number to be treated according to the protocol. The patient and the physician will decide the cNFs 
to be treated.  
6.5.1 
Inclusion criteria and selection 
• 
Males and females 18 years of age or older. 
• 
Have a clinical diagnosis of NF1 based on the most recent published criteria (35) (Appendix 
2) 
• 
Patients must be seeking active treatment for cNF. 
• 
Patients must have ≥ 5 cNF that are visible and measure a minimum of 2 mm in size. These 
must be in areas amenable to treatment and surveillance. 
• 
Patients with Fitzpatrick Type I to VI skin-type 
• 
Able and willing to comply with all visits, treatments, evaluations, schedules, and 
requirements. 
• 
Patients shall have received oral and written study information, accepted participation and 
signed the informed consent document. 
• 
Patients who are willing and mentally and physically capable to understand and follow the 
treatment and follow-up schedule including post-treatment care instructions. 
• 
Patients who are willing to have photographs and images taken of the treated lesions to be 
used anonymously or coded in evaluations and publications. 

 
 
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6.5.2 
Exclusion criteria for participant selection (non-inclusion criteria) 
• 
Patients who are undergoing other treatment modalities or investigational agents for their 
cNF lesions. 
• 
Individuals who cannot give informed consent or adhere to study schedule. 
• 
Patients who are actively tanning during the course of the study. 
• 
Patients with adverse reactions to compounds of any external agent in use. 
• 
Patients with known allergy to injectable anesthetics (relevant for biopsy only). 
• 
Patients with any condition which, in the Investigator’s opinion, would make it unsafe (for the 
participant or study personnel) to treat the participant as part of this research study. 
• 
Patients, where target treatment may cause the acoustic beam to enter the eye. 
• 
Pregnant females, due to possible discomfort with the procedure even though the procedure 
is localized and there is no new drug. 
• 
Patients with tendency for keloid and hypertrophic scar formation. 
• 
Patients with impaired wound healing. 
• 
Patients with any other acute or chronic condition which, in the opinion of the investigator, 
could interfere with the conduct of the study. 
6.5.3 
Criteria and procedures for participant withdrawal or lost to follow up 
• 
Exclusion criteria identical to non-inclusion criteria, however, appearing during the study. 
• 
Patients acquiring disease of higher priority contraindicating their further participation or 
making participation not possible in practice. 
• 
Patients' decision to leave the trial, irrespective of reason. 
• 
Patients' non-adherence to the study schedule. 
• 
Unforeseen hazards or significant clinical adverse events making continuation of the study 
not ethical. 
6.5.4 
Withdrawal from the Study 
Each participant has the right to withdraw from the study at any time without prejudice. The 
investigator may discontinue any participant’s participation for any reason, including failure to comply 
with the protocol (as judged by the investigator such as compliance below 80%, failure to maintain 
appointments, etc.). 
Should a participant withdraw from the study, the reason(s) must be stated on the case report form, 
and a final evaluation of the participant should be performed. 
Patients who go off treatment must be followed for AEs for at least 30 days from treatment or biopsy. 
Reason for withdrawal from study is documented in the case report form. Withdrawals after HIFU 
treatment and before the 3-month visit are replaced and not included in the analysis of results (but 
registered as dropouts). Withdrawals after 3 months are replaced on investigators discretion. 
6.5.5 
Study Termination Criteria 
The study may be terminated for multiple potential reasons. Some of these reasons include: 
• 
Majority of participants experience severe and unbearable pain, on investigator’s decision 
based on a minimum of 3 participants. 

 
 
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• 
Unbearable immediate wheal and flare reaction with severe itch and swelling and urticaria 
disseminating to larger skin areas experienced by a minimum 3 patients, on investigator’s 
decision as indicated above. 
• 
Any systematic (general or local) acute response to the treatment deemed by the investigator 
to be a clinically significantly different clinical or psychological reaction or adverse event than 
expected.  
• 
The efficacy of the treatment is in the majority of participants at the 3-month interim 
assessment (visit 4) deemed to be unacceptably poor by the investigator or the sponsor. 
Unacceptable efficacy is defined as cure rate below 20% at the 3-month visit, based on a 
sample of minimum 8 participants in each group of the study. 
6.5.6 
Participant enrollment and handling 
Every effort will be made to arrange routine clinical follow-up for all participants, independent of how 
many study-associated visits they have completed.  
Enrolment into study will take place at the two participating hospital departments (Department of 
Dermatology, Bispebjerg University Hospital and Department of Dermatology and Venereology, 
Sahlgrenska University Hospital).  
 
The total duration of the study from enrolment of first participant to end of data collection is expected 
to be 1.5 years. 
Expected total duration of each participants’ participation is 9 months, with milestone evaluation after 
every 3 months. 
In total 20 participants will be enrolled (target enrolment is 10 participants at Department of 
Dermatology, Bispebjerg University Hospital and 10 participants at Department of Dermatology and 
Venereology, Sahlgrenska University Hospital). Enrolment is not stratified. Limits can be changed 
based on the coordinating investigators discretion if recruitment to one study group turns out being 
not possible or according to experience unrealistic. Enrolment is based on participants remitted to 
the centers for diagnosis and treatment of NF1. Participants with urgent treatment need are not 
enrolled. 
Participants of the study continue their course and normal registration in the hospital department. 
Each study visit is registered and documented in the hospital file parallel to registrations in the case 
reports of the study. 
Histology taken at the Swedish center will be labeled using the study codes which do not contain the 
personal identification number of the study person. The samples will be used only for research 
purpose and not for patients care and treatment. The samples will be analyzed separately by a 
pathologist and the PI in Sweden. After the analyses the samples will be stored in Biobank Väst in 
Sahlgrenska Universitetssjukhuset, 
6.6 Procedures 
The study will be conducted according to the requirements of Good Clinical Practice (GCP) as per 
ISO 14155:2020. 
The study personnel will inform the participants verbally and in a participant information document 
in the respective local language on the experimental procedure to be utilized, and assure the 
participants that their decision regarding participation in the study will have no bearing on the quality 

 
 
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of medical care received, and that their decision whether to participate in the study is strictly 
voluntary. 
During the initial interview, the participants will be assured that they are free to change their mind 
and will be allowed to participate in the study or withdraw from the study with no adverse effect on 
their standard medical care. Participants will have at least 24 hours to consider their consider their 
participation. 
The study personnel will obtain written Informed Consent prior to the participants’ participation in any 
study procedure. 
The investigational device is described in detail in the investigator’s brochure. The device is used in 
the study as described in the brochure. The two devices used in the study are identical and validated 
to be so by the equipment supplier. 
The standard choice of hand piece will typically be those with a focal point depth of 1.8 or 2.3 mm. 
The hand piece depth shall match the estimated thickness of the tumor best possible. The hand 
piece with focal depth 1.8 mm will be preferred for treatment on the extremities, where the skin is 
known to be relatively thin. For treatments on the trunk, where skin is known to be thicker, the hand 
piece with focal depth of 2.3 mm will be preferred. The choice will be guided by tumor size and 
thickness of the overlying skin, location, and the investigator can decide to use hand piece best 
suited for the case. The tumor shall preferably be exposed to ultrasound down to the bottom of the 
tumor, but not so deep as to increase risk of post-treatment scarring. The selected hand piece 
applied for each tumor is documented in the CRF. 
Prior to the HIFU treatment, the selected area will be sterilized with an alcohol swab and the selected 
area will be outlined with a pen where necessary. The tumor will be documented by a photo, repeated 
immediately post treatment, and at the planned visits after 1-week, 3 months and 9 months. 
HIFU treatment will primarily be administered with shot-duration of 150 milliseconds, as this has 
been demonstrated in an animal safety study and in human treatments to be effective, and at the 
same time sufficiently short to avoid disturbing effects from movement of the hand piece during 
energy delivery. 
HIFU power will be selected dependent on treatment site and skin response in initial test shots 
applied to the tumor. Power levels between 4 to 9 W acoustic power is the range of operation of the 
device. The acoustic energy dosage will, thus, be in the range of 0.6 to 1.3 J/shot. 
On-going ex-vivo results will be used to validate these treatment guidelines and potentially adjust 
accordingly. Adjustments, if relevant, will not exceed existing certified clinical settings or existing 
standard limitations of the device.  
The investigator can decrease or increase the energy level guided by the visual assessment of the 
observed treatment dose effect in the tumor. A HIFU-dose shall produce an immediate visible 
whitening or contraction of a circular area sized about 1-2 mm around the target center. This is 
observed through the integrated dermoscope allowing direct control of the treated field. Dosing is 
applied as shoulder-by-shoulder covering the entire tumor and 2 – 3 mm of perilesional skin, 
immediately around the tumor, with 1-2 mm between each dose. The investigator can choose to 
treat the surrounding (non-thickened) skin with lower energy to save normal tissue and minimize the 
post-treatment wound. 

 
 
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The treatment of a cNF, which is in the range of 2 - 12 mm in horizontal width, is expected to require 
approximately 25 - 50 shots spaced nearby each other, “shoulder-by-shoulder”. Elapse between 
shots is some 2 - 3 seconds. The total direct time for a treatment is, thus, estimated to be not more 
than 5 minutes, including small pauses to monitor the treatment effect and the need of adjustment 
of device setting. The total time for the treatment session is estimated to be in the range 20 – 40 
min. 
The equipment supplier is responsible for control and maintenance of the investigational device 
during the study including check of the energy output. 
The equipment supplier has a third device in stock available for the study in case of instrument 
breakdown of a study device during study. 
Clinical recording of the study includes clinical assessment of healing and therapeutic effect on the 
tumors. Such assessment follows clinical criteria described in textbooks, guidelines and clinical 
consensus in the specialty of dermatology. Prior to study, the two investigator sites have coordinated 
their clinical assessment method. 
The chosen 9-month total study period is considered acceptable for observation of tolerance and 
curative effect of HIFU for cNF with observation of primary therapeutic effect after 3 months. 
Assessments includes monitoring of the wound healing phase, observation of late recurrences, and 
observation of scarring. 
The study includes no follow up medication, but the study can involve use of wound healing dressings 
and other remedies in the wound healing phase on investigators individual recommendation. The 
study involves no standard recommendation on post treatment care, which is anticipated to be 
simple. 
After the study participants are followed in the respective hospital departments according to their 
needs and the department routines. 
Participants who drop out of the study before the 3-month visit voluntarily or for non-medical reasons, 
including non-adherence, will be replaced by new participant(s). Withdrawals after 3 months are 
replaced on investigators discretion with considerations to the impact of the overall investigation 
schedule. 
Patient with incomplete cure documented by ultrasound imaging assessment or biopsy and histology 
after 3 months or subsequent control visit (indicated by clinical suspicion of reoccurrence) will be 
offered a second treatment based on the standard treatment offered by the center. The specific 
treatment will be decided by the investigator and the patient based on the extent and severity of the 
recurrence. 
The study will conclude when all participants have completed the 9-month study period, when all 
data are on file, and the statistical analysis is concluded. Interim analysis of data collected after 3 
months and later is allowed, per investigator’s decision.  
If the study design is deemed inferior, the protocol can be amended. 
The study produces no physical test samples relevant for purposes outside the study. 
 

 
 
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6.7 Monitoring plan 
The study will be monitored separately in the two centers. In Denmark, it is intended that the study 
is monitored by GCP-enheden ved Københavns Universitetshospital. In Sweden, the study is 
intended to be monitored by Gothia Forum for Clinical Studies. Both are units serving the respective 
regional healthcare researchers. 
As the monitoring unit in the region of the sponsor’s center, the Danish GCP unit will have the 
responsibility to coordinate a shared monitoring plan, where each party monitors the investigation 
center. The monitoring plan will be made with due consideration to the centers designated tasks and 
reflect considerations to specific risks in the tasks performed.  
An overview of monitoring activities and focus-areas are given in Table 4. 
Table 4. Monitoring activities in the two monitoring units used by the investigation centers. 
Investigation activity 
Stage /  
Visit 
GCP Unit KU, 
DK 
Gothia Forum, 
SE 
Monitoring plan 
Before 
X 
(x) 
Initiation of study 
Before 
X 
X 
Recruitment 
Before 
X 
X 
Screening and inclusion/exclusion 
Visit 1 
X 
X 
Selection and documentation of lesions for 
treatment 
Visit 2 
X 
X 
HIFU Treatment 
Visit 2 
X 
X 
Post treatment documentation and evaluation by 
investigator and patient 
Visit 2 
X 
X 
Follow-up visits with documentation and 
evaluation by investigator and patient 
Visit 3 - 6 
X 
X 
Ultrasound scanning – Procedure and analysis 
from measurements 
Visit 2 – 6 
X 
 
Biopsies – Procedure and analysis from 
pathological report 
Visit 2 – 4 
 
X 
CRF entry and REDcap 
Visit 1 - 6 
X 
X 
Statistical analysis 
After 
 
X 
Study Reporting  
After 
X 
 
Close of study – Archive readiness 
After 
X 
X 
 
 

 
 
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7 Statistical design and analysis 
The number of patients recruited for participation in the study will be balanced with due consideration 
to ethical aspects and the statistical background for conclusive statements regarding the primary 
endpoint. 
As mentioned above, the primary endpoint of the study is to document the tolerability and safety 
profile of the treatment. As the main measurable endpoint, the grade of overall adverse effects 
following treatment is best suited for evaluation of participants in the study. The measurement of this 
endpoint is done subjectively by the investigator according to a conventional grading system, and is 
measured directly after treatment and at all subsequent control visits. The input template for 
measurement is illustrated in Fig. 7. 
 
Investigator overall scoring of treatment side effect severity (one X) 
Absent / Normal 
Mild 
Moderate 
Severe 
Life-threatening 
or disabling  
Death 
Grade 0 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Fig 7. The primary end-point of the study is the investigator’s grading of adverse effects following 
treatment as measured directly after treatment and in all subsequent patient follow-up visits.  
 
According to the defined endpoint, the minimum number of patients must therefore be calculated to 
support a sufficiently high power for the statement that not more than 30% of patients will experience 
an overall side-effect of Grade 3 or above based on Common Terminology Criteria for Adverse 
Events (CTCAE), U.S. Department of health and human services, Ver 5.0, Nov 27, 2017. 
Secondary endpoints regarding efficacy, feasibility etc. are also measured and analyzed throughout 
the study, and form important data for analysis, but are not included in the calculation of the minimum 
number of patients. 
To obtain an initial indication of the necessary number of participants needed in the study, a simple 
estimation based on the conventional calculation of sample size to estimate a proportion or apparent 
prevalence with specified precision can be used. 
A simple binary outcome is considered: 
• 
Accept: Participants have Grade 2 side effects or lower 
• 
Reject: Participants have Grade 3 side effects or higher 
Based on substantial previous clinical experience with the study device (used for other skin 
treatments), severe side effects and adverse events are very rare, and an estimated proportion of 
90% having Grade 2 or lower side-effects is therefore a reasonable starting point for the calculation 
(P=0.90). 
As the measurement of side-effects by the investigator is subjective and with a quite course 
resolution, a total error can be set to 30% (e = ±0.15), roughly reflecting the interval between the six 
possible gradings. A standard confidence interval of 95% can be used (Z = 1.96). 

 
 
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At these inputs, the minimum population for the investigation can be calculated as follows: 
N = (Z2 x P x (1 - P))/e2 = (1.962x 0.90 x (1 – 0.90)/0.152) = 15.37 
At 16 participants in the study, it is therefore possible to say with 95% confidence that the results of 
treatments will fall within the acceptable group. The minimum number of participants at various other 
proportions is shown in Table 2. 
 
Table 2. Calculated minimum population in an investigation for obtaining statistical significance at a cure 
rate of p and with a error of ±15%  
 
 
In a second step, power curves can be calculated against an alternative base-line of equal 
proportions having acceptable and rejectable grades of side-effects (the null hypothesis). 
Fig 8. shows the power curve for the study containing the above minimum 16 participants as a 
function of the actual obtained passed AE-profiles. At the hypothesis of 90% having an acceptable 
AE-profile after HIFU treatment the power is calculated to be 71%. 
Similarly, curves correlating the number of necessary participants at fixed power-levels of 80% and 
90% at the hypothesized 90% having an acceptable AE-profile after HIFU treatment can be 
calculated. As shown in Fig. 9, the study will require 20 participants if a power of 80% is needed, 
while 26 participants will be needed to reach a power of 90%. 
 
 
Fig. 8. Power curve showing the probability of being able to detect difference between HIFU treatment 
with 90% passed AE profile against a baseline treatment with 50% acceptable proportion. At the projected 
succes rate of 90%, the difference in cure rate can be detected with a 95% confidence level and power of 
above 71% if the population in the investigation is 16 participants. 
p
0,95
0,90
0,85
0,80
0,75
0,70
0,65
0,60
0,55
0,50
N
9
16
22
28
33
36
39
41
43
43

 
 
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Fig. 9. Power curves showing the necessary population to obtain power levels of 80% and 90%. At the 
projected acceptable AE-profile of 90%, the hypothesis can be confirmed with a power of 80% probability 
if the population is 20 participants and a poer of 90% if the population in the investigation is 26 participants. 
 
In conclusion, the statistical analysis shows that participation as low as 16 patients will give an 
acceptable power, provided the AE profile is in the expected range. At worse AE-profiles, or 
requirements for a higher power-level in comparison with alternatives, the required population size 
naturally increases, and populations above 20 will typically needed. 
In consideration of the status as an initial tolerability and feasibility study performed on a relatively 
young population group with low expected drop-out, it is considered that a total of 20 participants 
distributed among the two centers will be a well-balanced population taking ethical, clinical and 
practical issues into due consideration. 
It should finally be mentioned that each participant will present a minimum of 4 tumors for treatment, 
and sometimes significantly more than this. The total number of treated tumors in the study will thus 
be high. Under certain assumptions, this allows for pooling of data on separate tumors during the 
data-analysis phase, rather than looking at combined reactions and results on participant-level only. 
The CRF in the study accommodates such individual data collection. 
 
8 Data management 
The study shall respect all legal requirements regarding data protection of the participant, with the 
necessary exceptions specified in the study consent form. As a study conducted by two centers in 

 
 
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the EU, the overall framework of the European Regulation on General Data Protection Regulation 
2016/679 (GDPR) with its respective national adaptations will govern data management.  
In Denmark, all information about participants is subjected to “Lov om behandling af 
personoplysninger” and “Sundhedsloven”, and these will be complied. The approval from Danish 
Data Proctection Agency (dataanmeldelse i Region Hovedstaden) will be applied. 
In Sweden, all information about participants is particpanted to The General Data Protection 
Regulation of the EU, and “25 kap. 11 § 5 p. offentlighets- och sekretesslagen (2009:400) (OSL)”, 
and these will be complied. The approval from Etikprövningsmyndigheten will be applied. 
Each participant will be informed that representatives from the respective national data protection 
agency and Medicines Agency may inspect their medical journals and trial records, in all 
confidentiality. The requirements to ensure anonymity of data, data security and confidentiality of 
data will be explained to the participants and complied. 
All information will be treated with strict confidentiality and stored as confidential material according 
to the respective national regulations. Only the principal- and coordinating investigators will have 
access to the information. 
Study results will be reported in anonymous form. The investigator will keep identification lists of all 
participants. This list will include full name and social security number. 
The recorded data will be kept in a Case Report Form and an individual journal will be created for 
the investigation. The information will only be available for inspection for authorized representatives 
from the relevant authorities upon request (national GCP Unit, Data Protecting Agency, Medicines 
Authority). 
During the investigation, paper-based CRF will be used. All information according to protocol and 
collected during the study must be entered in the appropriate field of the CRF. The investigator, or 
designated representative, should complete the appropriate CRF fields as soon as possible after 
information is collected. The information must match the information that exists as source documents 
in the clinic chart, hospital chart, and/or investigator’s files. An explanation should be given for all 
missing data. REDCap will be used for storage of such information afterwards. 
The investigators in each center will retain investigational records, copies of CRF and source 
documentation for the maximum period required by the regulatory authorities. 
The center in Denmark will be appointed as data-processor. Only pseudonymized data will be 
transmitted from the center in Sweden to Denmark for combined processing and reporting. A 
cooperation agreement including encrypting of the files and relevant provisions for data transfer and 
management will be made between the two centers to ensure that transfer of such data is in 
compliance with GDPR and the legislative requirements in both countries. 
It is the coordinating and principal investigators at each center responsibility to assure the accurate 
completion, review, and approval of all CRFs, and the timely completion and submission of all 
adverse event forms. 
In the case of a personal data breach, the relevant investigator(s) must without undue delay and, 
where feasible no later than 72 hours after having become aware of it, notify the personal data 
breach to the respective national Data Protection Agency, unless the personal data breach is unlikely 
to present a risk to the affected individuals. 

 
 
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Reporting must be done as per the national guidelines, e.g. via electronic log-in to the relevant 
portals. 
9 Amendments to the CIP 
 
If the sponsor intends to introduce modifications to a clinical investigation that are likely to have a 
substantial impact on the safety, health or rights of the subjects or on the robustness or reliability of 
the clinical data generated by the investigation, it shall notify, within one week, the relevant national 
authorities in Denmark and Sweden of the reasons for and the nature of those modifications.  
The sponsor shall include an updated version of the relevant documentation as part of the 
notification. Changes to the relevant documentation shall be clearly identifiable.  
The relevant authorities in Denmark and Sweden shall assess any substantial modification to the 
clinical investigation.  
The sponsor may implement the modifications at the earliest 38 days after the notification unless:  
a) A medical authority in Denmark or Sweden has notified the sponsor of its refusal based on 
the grounds referred to in MDR Article 71(4) or on considerations of public health, subject 
and user safety or health, of public policy, or  
b) An ethics committee in Denmark or Sweden has issued a negative opinion in relation to the 
substantial modification to the clinical investigation, which, in accordance with national law, 
is valid for that entire country.  
The relevant authorities concerned may extend the period for decision by a further seven days, for 
the purpose of consulting with experts. 
 
 
10 Deviations from clinical investigation plan 
Deviations to the CIP are not allowed unless they affect the subjects’ rights, safety and well-being, 
or affect the scientific integrity of the investigation.  
Deviations needed to protect the rights, safety and well-being of human subjects under emergency 
circumstances may proceed without prior approval of the sponsor and the relevant authorities.  
Deviations, which are systematic, necessary, and significant to the study, i.e. affect the subjects’ 
rights, safety and well-being, require a protocol amendment with submission and approval from the 
relevant authorities.  
Insignificant, minor and unsystematic deviations to the CIP incurred to maintain the scientific integrity 
of the investigation, need notification in the case report only. 
All deviations must be documented and reported to the sponsor, and must be included in the case 
report of the day of the visit or the deviation. All deviations shall made available for study monitor’s 
inspection.  

 
 
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11 Device accountability 
The sponsor is responsible for registration of the accounts of the device, properly documented. 
Delivery, placement and return of device has no special risk of hazard and needs no special 
procedure. No special risk of fire or contamination is noted. 
 
12 Statements of compliance 
The sponsor carries responsibility that statements signed by the participating centers/investigators 
on the fulfilment of the study in accordance with the Declaration of Helsinki, regional and national 
regulations and any relevant authority or legal requirement. The statements are kept in a study 
master file. 
The equipment supplier shall provide insurance of hazards and accidents related to the use of the 
device in the study. Hazards and accidents independent of the device and related to the hospital 
setting are covered by the standard insurance of the hospital. Any hazard or accident in a participant 
is reported in the participants’ patient file belonging to the hospital, and as adverse event in the case 
report. 
 
13 Informed consent process 
Candidates for the investigation are hospital patients referred to the participating departments for a 
cNF tumor. They all are registered in the standard electronic hospital files. 
Potential candidates are informed orally and in writing about the investigation using the standard 
information of the study submitted to Lægemiddelstyrelsen and the Danish National Committee for 
Health Research Ethics. 
Oral information is provided at an information-meeting with the participating investigator(s). The 
candidate will be informed in advance that he/she is allowed to bring an assessor for the meeting. 
The meeting will be held in a room that will only be used for this purpose to avoid unnecessary 
disturbances. The written information includes investigation participation document as well as 
relevant information folders from the respective national authorities. In Denmark this includes the 
document "Dine rettigheder som forsøgsperson" from “De Videnskabsetiske Medicinske Komiteer 
(VMK)”. In Sweden this includes printed information for research persons which will be accepted by 
Etikprövningsmyndigheten pror to the study. 
The informed consent form will include all the elements that are required according to the respective 
national legislation. If the participant wishes to participate, a new appointment will be booked where 
the doctor and the participant sign the consent. The new appointment and time of reflection will be 
within 2-7 days after the first appointment (allowing more than the required 24 hours of reflection 
time), but if the patient needs more time of reflection this can also be accommodated. If the patient 
wishes to sign the content at the first appointment this is also acceptable. If the patient fulfills all in- 
and exclusion criteria the patient will be included. 

 
 
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After the information-meeting, all active participants will be updated with any new and important 
information or change in the study that can have influence on the participants’ willingness to 
participate or safety. The approved documents will be updated accordingly. 
Oral information is repeated on the inclusion visit (visit 1 in the CRF), with opportunity for the patient 
to ask questions. The consent form is signed after the patient has declared the full understanding of 
the information and the study concluded as acceptance to enter study. In the oral information 
individual aspects of special relevance to the participant shall be emphasized. 
Participants who are not able to understand the information cannot be included in the study. The 
signed and qualified acceptance of the participant to volunteer for the study is filed in the case report 
form book. 
 
14 Adverse events, adverse device effects, and device deficiency. 
14.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence, unintended disease or injury or any 
untoward clinical signs, including an abnormal laboratory finding, in subjects, users or other persons, 
in the context of a clinical investigation, whether or not related to the investigational device. 
14.2 Serious Adverse Events  
A serious adverse event (SAE) is any event that led to any of the following: 
a) death, 
b) serious deterioration in the health of the subject, that resulted in any of the following: 
I. 
life-threatening illness or injury, 
II. 
permanent impairment of a body structure or a body function, 
III. 
hospitalization or prolongation of patient hospitalization, 
IV. 
medical or surgical intervention to prevent life-threatening illness or injury or 
permanent impairment to a body structure or a body function,  
V. 
chronic disease,  
c) foetal distress, foetal death or a congenital physical or mental impairment or birth defect 
14.3 Device deficiency 
A device deficient is any inadequacy in the identity, quality, durability, reliability, safety or 
performance of an investigational device, including malfunction, use errors or inadequacy in 
information supplied by the manufacturer. 
14.4 Reporting to national authorities 
The sponsor shall report, without delay to the respective national Medicines Agency all of the 
following: 
a) any SAE that has a causal relationship with the investigational device or the investigation 
procedure or where such causal relationship is reasonably possible. 

 
 
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b) any device deficiency that might have led to a SAE if appropriate action had not been taken, 
intervention had not occurred, or circumstances had been less fortunate. 
c) any new findings in relation to any event referred to in points (a) and (b). 
The period for reporting shall take account of the severity of the event. Where necessary to ensure 
timely reporting, the sponsor may submit an initial report that is incomplete followed up by a complete 
report. 
Analysis of the reporting requirements for AE’s and SAE’s, i.e. whether reporting should be sent to 
the respective medical authority, will be conducted as per the guidelines given by the Medical Device 
Coordination Group (MDCG) in “MDCG 2020-10 Guidance on safety reporting in clinical 
investigations”. Reporting will be done electronically using the templates included in the appendix to 
this guideline (MDCG 2020-10/2). 
Records and reports of events shall include accurate and relevant information on type of event, date, 
treatment, resolution, assessment of seriousness and relationship to the investigational device and 
the whole procedure. 
The investigational device applies ultrasound generated focused heat to the outer skin, being the 
mode of action. Unexpected stronger burn than anticipated according to instrument setting is a 
potential adverse event, which may result in more severe necrosis with delayed wound healing. 
However, such event is due to the construction of the transducer and the hand piece, and in any 
case limited to the skin, and therefore primarily a local tolerance issue. 
Pain associated with the treatment is foreseeable and an expected adverse event, however, unlikely 
to be significantly worse than pain associated with other instrumental treatments of skin in general 
use such as surgical interventions, laser treatment and photodynamic treatment of skin cancer. 
The two study centers are hospital departments with immediate access to emergency treatments. 
 
15 Vulnerable population 
The study does not include identified vulnerable participants, see inclusion and exclusion criteria. 
 
16 Suspension or premature termination of the clinical 
investigation 
The study is suspended if serious adverse events occur related to the investigational device, and 
the decision taken by the primary investigator and the sponsor to either terminate the study or 
continue the study depending of the clarification of the case. Continuation of study may require a 
protocol amendment. 
The study is unblinded and no issue re. premature unblinding is relevant. 
Participants having device-related adverse events are followed up upon investigator’s discretion with 
control and medical treatment as required in the individual case. 

 
 
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17 Publication policy 
The study will be registered in the European registry (www.clinicaltrials.gov) for ongoing clinical trials. 
The result of the study shall be published in a peer-reviewed journal with the consent of all 
participating investigators, regardless if the overall results of the study is positive or negative. 
The research publication shall be submitted to a journal within 6 months after the final study report 
is available. 
 
 
 

 
 
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