Improving Survival of COlorectal LIver Metastases by RFA-mediated Immunostimulation
Phase: NA
Conditions: Liver Metastasis Colon Cancer
Sponsor: University of Aarhus

Protocol 
Version 4 
01.11.2021 
 
1 
  
The ISCOLIM trial: Improving Survival of Colo-rectal Liver Metastases 
by RFA-mediated Immuno-stimulation 
 
 
Participating clinical and research centres: 
Department of Surgery, Aarhus University Hospital 
Department of Surgery, Rigshospitalet Copenhagen 
Department of Immunology, Aarhus University Hospital 
Department of Molecular Medicine, MOMA, Aarhus University 
 
Sponsor, Primary clinical investigator (PI) 
Professor Frank Mortensen 
Department of Surgery 
Section of hepato-pancreatico-biliary surgery 
Aarhus University Hospital  
Palle Juul-Jensens Boulevard 99 
8200 Aarhus, Denmark 
 
 
+45 7846 4512 
 
 
+45 2251 7296 
franmort@rm.dk 
 
Translational Investigator of Molecular Biology (TI) 
Professor Claus Lindbjerg Andersen 
Department of Molecular Medicine 
Aarhus University Hospital  
Palle Juul-Jensens Boulevard 99 
8200 Aarhus N, Denmark 
+45 7845 5319 
+45 2980 4321 
cla@clin.au.dk 
 
Translational Investigator of Immunology (TI)  
Associate Professor, Chairman, Bjarne Kuno Møller 
Head of the Blood Bank and Department of Immunology 
Aarhus University Hospital  
Palle Juul-Jensens Boulevard 99 
8200 Aarhus N, Denmark 
+45 78455000 
bjmoel@rm.dk 
 
 

Protocol 
Version 4 
01.11.2021 
 
2 
Trial administrative structure 
This is a sponsor-initiated study. Professor Frank Viborg Mortensen is the sponsor and primary 
coordinating investigator (PI). Professor Claus Lindbjerg Andersen is the coordinator of the trans-
lational analyses, and Associate Professor Bjarne Kuno Møller is the coordinator of the immuno-
logical analyses. The decision-making body of the study is the steering committee (SC), which 
will have representation from all participating departments. Professor Frank Mortensen is chair of 
the SC. 
 
Collaborators 
Aarhus University Hospital: 
Jakob Kirkegård, MD, Ph.D., Department of Surgery 
Anders Riegels Knudsen, Associate Professor, MD, Ph.D., Department of Surgery 
Andrea Lund, MD, Ph.D.-student, Department of Surgery 
Jens Erik Nielsen, MD, Ph.D. Department of Invasive Abdominal Radiology 
Nicklas Heine Staunstrup, External Associate Professor, M.Sc., Ph.D., Department of Immunol-
ogy 
Bjarne Kuno Møller, Associate Professor, MD, Head of the Blood Bank and Department of Im-
munology 
 
Department of Molecular Medicine, MOMA 
Claus Lindbjerg Andersen, professor, M.Sc., Ph.D., MOMA 
Mai-Britt Worm Ørntoft, MD, Ph.D., MOMA 
 
Rigshospitalet: 
Peter Nørgaard Larsen, MD, Department of Gastro-intestinal Surgery 
Nicolai Schultz, MD, Ph.D., Department of Gastro-intestinal Surgery 
Jens Georg Hillingsø, Consultant, MD, Ph.D., Head of department of Gastro-intestinal Surgery 
 
Regionernes Bio- og Genombank (RBGB) 
 

Protocol 
Version 4 
01.11.2021 
 
3 
Table of contents 
THE ISCOLIM TRIAL: IMPROVING SURVIVAL OF COLO-RECTAL LIVER METASTASES BY RFA-
MEDIATED IMMUNO-STIMULATION ................................................................................................. 1 
Collaborators ...................................................................................................................................................... 2 
TABLE OF CONTENTS .......................................................................................................................... 3 
STUDY SUMMARY ............................................................................................................................... 6 
1. INTRODUCTION .............................................................................................................................. 8 
2. OBJECTIVES ................................................................................................................................... 10 
2.1 Primary aim ................................................................................................................................................ 10 
2.2 Secondary aims ........................................................................................................................................... 11 
2.2.1 T-cell response to tumor development .......................................................................................................... 11 
2.2.2 ctDNA as biomarker of residual disease or recurrence ................................................................................. 11 
2.2.3 Gene expression signatures ........................................................................................................................... 12 
3. INVESTIGATIONAL PLAN ............................................................................................................... 12 
3.1. Overall study design ................................................................................................................................... 12 
3.2 Study timetable ........................................................................................................................................... 14 
3.3 Setting ......................................................................................................................................................... 15 
3.4 Sample size .................................................................................................................................................. 15 
3.5 Study feasibility ........................................................................................................................................... 15 
3.6 Inclusion and exclusion criteria for patients ............................................................................................... 15 
3.6.1 Inclusion criteria ........................................................................................................................................... 16 
3.6.2 Exclusion criteria .......................................................................................................................................... 16 
3.7 Time of inclusion and allocation procedure ................................................................................................ 16 
3.8 Procedures for patient inclusion and informed consent .............................................................................. 16 
3.9 Patient information and consent ................................................................................................................. 18 
3.10 Randomization .......................................................................................................................................... 18 
3.10.1 Study Flow .................................................................................................................................................. 18 
3.10.2 Baseline visit (-14 days from operation) ..................................................................................................... 19 

Protocol 
Version 4 
01.11.2021 
 
4 
3.10.3 Month 3 to 60 .............................................................................................................................................. 19 
3.11 Radio-frequency ablation (RFA) ............................................................................................................... 20 
3.12 Collection, handling, and biobanking of biological samples ...................................................................... 20 
3.13 Research biobank and biobank for future research .................................................................................. 21 
3.14 Molecular analyses .................................................................................................................................... 21 
3.14.1 CEA analysis ............................................................................................................................................... 21 
3.14.2 Immunological analyses .............................................................................................................................. 22 
3.14.3 ctDNA analyses ........................................................................................................................................... 22 
3.14.4 T-cell receptor analyses ............................................................................................................................... 23 
3.14.5 DNA sequencing ......................................................................................................................................... 23 
3.14.6 DNA Methylation analyses ......................................................................................................................... 23 
3.14.7 RNA sequencing ......................................................................................................................................... 24 
3.14.8 Data analyses ............................................................................................................................................... 24 
3.15 Collection of clinical information .............................................................................................................. 24 
3.16 Definitions of predictor variables, co-variates, and endpoints .................................................................. 25 
3.16.1 Primary endpoint ......................................................................................................................................... 25 
3.16.2 Secondary endpoints ................................................................................................................................... 25 
3.16.3 Safety Endpoints ......................................................................................................................................... 25 
3.16.4 Definition of endpoints ............................................................................................................................... 26 
3.17 Statistical analyses ..................................................................................................................................... 26 
3.17.1 Demographics and baseline data ................................................................................................................. 26 
3.17.2 Analysis of OS, TTCR, and TTMR ............................................................................................................ 26 
3.17.3 Analysis of Safety ....................................................................................................................................... 27 
4.1 Quality Control ........................................................................................................................................... 27 
5. PROCESSING OF PERSONAL DATA IN THE ISCOLIM TRIAL STUDY ............................................... 29 
6.1. RFA ............................................................................................................................................................ 30 
6.2. Blood samples ............................................................................................................................................. 31 
6.3 Tissue samples ............................................................................................................................................. 31 
6.4 CT scans ...................................................................................................................................................... 31 
6.5 
Genomic sequence analysis .................................................................................................................. 32 
7. PUBLICATION AND REPORTING FROM THE TRIAL ....................................................................... 33 
8. ECONOMY ..................................................................................................................................... 33 
8.1 Remuneration to participants ..................................................................................................................... 33 
Deleted: 
Deleted: 
Deleted: 
Deleted: 
Deleted: 2
Deleted: 3
Deleted: 3
Deleted: 3
Deleted: 3
Deleted: 3
Deleted: 3
Deleted: 3

Protocol 
Version 4 
01.11.2021 
 
5 
8.2 Patient compensation scheme (patienterstatningen) ................................................................................... 33 
 
 
 
 
 
Deleted: 3

Protocol 
Version 4 
01.11.2021 
 
6 
Study summary 
Title: 
Improving Survival of Colo-rectal Liver Metastases by RFA-
mediated Immuno-stimulation 
Acronym: 
The ISCOLIM trial: Improving Survival of COlo-rectal LIver 
Metastases by RFA-mediated Immuno-stimulation 
Principal investigator: 
Frank Viborg Mortensen, Aarhus University Hospital 
Funding organizations: 
Novo Nordisk Fonden  
Neye Fonden 
Study subjects: 
Patients with colo-rectal liver metastases (CRLM) 
Study design: 
Multi-centre Randomized Controlled Trial 
Aim: 
1. To examine the effect of radio-frequency ablation (RFA) 
on the immune system in patients undergoing curative-in-
tent surgery for CRLM. 
2. To characterize whether alterations to the peripheral T-cell 
receptor repertoire during the disease course reflect im-
mune cell recognition of cancer neoantigens. 
3. To examine the effect of RFA-mediated immune-stimula-
tion on survival in patients undergoing curative-intent sur-
gery for CRLM. 
4. To monitor disease recurrence with circulating cell-free   
DNA (cfDNA) biomarkers, specific for CRLM. 
Endpoints:  
1. Overall and disease-free survival for CRLM patients. 
2. Describe effect of innate and adaptive immune system on 
survival. 
3. Lead time of cfDNA biomarkers in prediction of recurrence 
compared to conventional imaging technique (CT scans). 
4. Description and characterization of T-cell receptor changes 
on a molecular level. 
Intervention: 
Intervention arm: RFA combined with liver surgery. 
Standard-of-care arm: liver surgery alone. 
Number of Subjects: 
220 patients (110 in each arm) enrolled consecutively. 
Selection Criteria: 
Inclusion: 
1. Patients diagnosed with metachronous or synchronous 
CRLM planned for liver resection 
2. At least one tumor with size ³3 cm 
3. Performance status 0-1 
4. Age ³18 years 
Exclusion: 
1. Liver cirrhosis 

Protocol 
Version 4 
01.11.2021 
 
7 
2. Non-curative extrahepatic metastases 
3. Other cancer within 5 years prior to CRLM diagnosis 
4. Earlier RFA treatment 
5. Patients who are unlikely to comply with the protocol 
Duration of study:  
The study duration is expected to be 18 months for subject re-
cruitment and additional 5 years of follow-up. Interim data 
analyses will be performed after inclusion of the first ten pa-
tients in each arm (expectantly at 6 months), and then after 12 
and 24 months. The study will commence on September 1, 
2021. 
Statistics: 
Cox proportional hazards model will be used to compare sur-
vival in the two arms. 
Power calculations: 
Assuming that the 1-year survival rate is 90% in the experi-
mental arm and 75% in the control arm, at a 0.05 significance 
level with a desired power of 80%, 100 patients are needed in 
each arm (200 in total). To account for an expected loss-to-fol-
low up of ~10%, 220 patients (110 in each arm) are included.  
 
 
 

Protocol 
Version 4 
01.11.2021 
 
8 
1. Introduction 
In Denmark, colo-rectal cancer (CRC) represents a significant health burden with ~5,000 new 
cases and ~2000 CRC related deaths in 2018(1). Death of CRC is primarily associated to distant 
metastases occurring at the time of diagnosis (synchronous) or appearing later in the course of the 
disease (metachronous), and those are most frequently found in the liver.  
Each year 1,600 Danish individuals are diagnosed with colo-rectal cancer liver metastases 
(CRLM)(2). For these patients, curative treatment options have evolved dramatically over the last 
10-20 years(3) and includes liver surgery, radio-frequency ablation (RFA), radiation therapy, and 
chemotherapy, and the relative 5-year survival approaches 50-60%(4-8). However, if CRLM me-
tastases are detected too late for intended curative treatment, the patients instead receive palliative 
chemotherapy or best-supportive care, and the 5-year survival rate decreases to <10%(4-6, 8). As 
the ability to offer curative treatment is influenced by the number of metastatic sites and the size 
of the metastases, early detection of CRLM is of paramount value. 
Minimally invasive blood-based analysis of circulating tumor DNA (ctDNA) in CRC has been 
extensively studied in recent years and has been shown to increase especially in metastatic CRC(9-
11). It is also a tool which potentially can identify microscopic cancer early(12-14). The ctDNA 
analyses is based on the finding that cancer cells shed DNA fragments into the blood(15). The 
half-life of ctDNA is less than two hours(16) so cancer-free patients will expectantly have no re-
maining ctDNA in the blood during follow-up after CRLM treatment, while patients with residual 
disease or recurrence are likely to be ctDNA positive. In follow-up studies of patients with locally 
advanced CRC, it has been shown that the risk of relapse in patients with postoperative ctDNA 
was close to 100%, while the risk of relapse in ctDNA negative patients was as low as 10% 
(HR=37.66; 95% CI, 4.23 to 335.49; P<0.0001)(13, 14, 17). Therefore, ctDNA could be a predic-
tor for either residual CRLM or CRLM recurrence, and followingly guide treatment decisions. 
Surgical liver resection remains state-of-the-art for treatment of CRLM. However, liver resection 
can only be performed if patients have a good performance status, and when at least 30% of the 
liver parenchyma can be preserved, which is unfortunately only possible in 25% of the patients(3). 
As an alternative, RFA is a low-risk procedure that can be performed safely in most patients, in-
cluding those with a poor performance status. By thermal ablation, RFA causes a localized coag-
ulation necrosis in the targeted tumor tissue: when the necrosis affects all cancer cells, and no 

Protocol 
Version 4 
01.11.2021 
 
9 
viable cancer cells remain, the treatment is curative. RFA is a parenchymal-sparing treatment for 
hepatic malignancies including CRLM and is particularly effective for tumors <3 cm(18, 19). For 
multiple or larger tumors it can be combined with liver resection, as a parenchymal-sparing strat-
egy that couple effective thermal ablation of small tumors with surgical resection of the larger 
tumors, for which ablation is less effective (20). 
Recently, significant attention has been given towards understanding the immune system’s role in 
cancer development, metastasis, and response to therapy. Here, especially the adaptive immune 
system is paramount, as it recognizes tumor neoantigens with which to eradicate cancer cells. Fur-
ther, the microenvironment and supportive stroma surrounding the tumor actively participates in 
the tumor development over time(21). Thus, it is to be expected that the immune response towards 
CRLM will change during the disease course in response to treatment, recurrence, or remission, 
and that it may be prognostic for the clinical outcome, as well as predictive for the response to a 
given curative treatment. As an example of the effect of the immune system on cancer develop-
ment, it has been shown that colon tumors with abundant infiltration from cytotoxic T lymphocytes 
(CTL’s) and NK cells have better prognosis than the tumors, which lack such immune re-
sponse(22). Another example is when the patient’s adaptive immune system is activated by tumor 
vaccines. A tumor vaccine functions by activating the patients’ own adaptive immune system 
against the cancer(23) and thereby facilitates tumor death by T-cell mediated apoptosis, and can 
be used as immune therapy in oncological treatment strategies(24). 
During RFA, necrosis of CRLM cells is induced, and their tumor antigens are released to the mi-
croenvironment, which has been shown to elicit an immune response in the patient(25). Therefore, 
the partial destruction of CRLM by RFA may possibly represent a method equal to a tumor vac-
cine, where tumor antigens are exposed and activates the host immune system and enhance immu-
nogenicity, thereby increasing overall survival. Nevertheless, the effect of RFA on immunogenic-
ity and the following impact on survival in patients with CRLM has never been examined in the 
clinic.  
 
 
 

Protocol 
Version 4 
01.11.2021 
 
10 
2. Objectives 
Overall, we aim to conduct a clinical randomized controlled trial (RCT) of patients with CRLM 
with a longitudinal follow-up. Within this RCT, we will study the effect of RFA and liver resection 
on immunogenicity and CRLM survival. The central hypothesis is that patients treated with RFA 
will elicit an increased immune response because of the RFA induced partial tumor necrosis that 
will increase overall survival and reduce cancer recurrence. We further hypothesize that 1) cancer 
vaccination using the ISCOLIM procedure will lead to alterations in the peripheral T-cell receptor 
(TCR) repertoire, reflecting immune cell recognition of cancer neoantigens released by the proce-
dure, 2) the induction of T-cell repertoire changes will predict the benefit of the ISOCOLIM pro-
cedure 3) characterizing the T-cell receptor repertoire provides an attractive solution with a poten-
tially amplified signal (due to T-cell expansion against the cancer) for assessing response, 4) the 
longitudinal durability of the T-cell repertoire changes will predict the durability of the clinical 
benefit of the ISCOLIM procedure, and 5) longitudinal monitoring of the T-cell receptor repertoire 
will facilitate early detection of relapse.  
Finally, we hypothesize that 1) ctDNA are a better diagnostic marker for residual disease after 
CRLM surgery than CT scans or CEA, that 2) ctDNA are a diagnostic marker of CRLM recurrence 
equal to CEA or CT scans, that 3) ctDNA are prognostic for overall survival, and 4) that ctDNA 
are predictive of response to oncological treatment better than CT scans.  
 
2.1 Primary aim 
We want to examine the effect of RFA-mediated immune-stimulation in patients undergoing cu-
rative-intent surgery for CRLM. We hypothesize that patients treated with RFA and liver resection 
have improved survival compared to patients treated only with liver resection, due to an increased 
immune response.  
Endpoints 
1. Increase in 3-year survival rate from 55% in the control arm to 75% in the experimental arm. 
2. Increase in 1-year survival rate from 75% in the control arm to 90% in the experimental arm. 
3. Statistically significant increase in recurrence free survival (RFS) in the experimental arm 
compared to the control arm. 

Protocol 
Version 4 
01.11.2021 
 
11 
2.2 Secondary aims 
2.2.1 T-cell response to tumor development 
We aim to characterize the longitudinal changes in T-cell in response to CRLM and the following 
surgical/RFA treatment. We hypothesize, that RFA treated patients will develop different immune 
cell characteristics than patients treated with liver surgery only. Further, we expect patients with 
recurrence during follow-up to present a change in immune response in comparison to patients 
with no recurrence. By T-cell receptor sequencing and RNA sequencing (RNA-seq), we will iden-
tify changes in the T-cell receptor repertoire.  
Endpoints 
1. Pre- to post treatment change in T-cell receptor repertoire 
2. Time to T-cell receptor recurrence (significant change in T-cell receptor repertoire) 
3. Durability of the change in T-cell receptor repertoire 
4. T-cell receptor repertoire change and correlation to RFS 
 
2.2.2 ctDNA as biomarker of residual disease or recurrence 
We will explore the use of ctDNA as a biomarker for prediction of incipient recurrence. The as-
sessment will be made both at milestone time-points and by serial analysis. We will explore if 
ctDNA can detect recurrence before standard-of-care CT imaging and before CEA and T-cell re-
ceptor repertoire analysis (lead-time analysis). We will explore if ctDNA can help guide clinical 
decision making at time-points where the CT-imaging result are indeterminate (~10% of all CT-
scans).  
Endpoints 
1. Time to radiological recurrence 
2. Time to ctDNA recurrence 
3. Time to CEA recurrence 
4. Time to T-cell-receptor recurrence 
5. Overall survival 
 

Protocol 
Version 4 
01.11.2021 
 
12 
2.2.3 Gene expression signatures 
With DNA and RNA sequencing, we will examine CRLM genes and their expression signatures. 
Further we will investigate changes in gene expression signatures associated to ISCOLIM treat-
ment response, CRLM recurrence, and overall survival.  
Endpoints  
1. Gene expression signatures and correlation to overall survival and RFS. 
 
3. Investigational plan 
3.1. Overall study design 
The present study will establish the sponsor-initiated, randomized prospective clinical trial IS-
COLIM. The RCT will investigate the benefit of RFA on survival for CRLM patients and charac-
terize changes in the immune system in response to treatment on a molecular level. Patients will 
be randomized within blocks of 10, such that an equal number are assigned to each treatment. 
Patients will be randomized to either the experimental arm or the control arm (Figure 1).  
 
          
                  
   
Figure 1. ISCOLIM Study design. Randomization for study I and II. 
 
Enrollment
Experimental arm
Control arm
Randomization
RFA
Surgery
Surgery
Surgery
Surgery
n:10
n:10
7 days
Study I
Enrollment
Experimental arm
Control arm
Randomization
RFA
Surgery
Surgery
Surgery
Surgery
n: 100
n: 100
Study II
Follow-up CT 
scan and blood
samples
Follow-up CT 
scan and blood
samples
30 minutes

Protocol 
Version 4 
01.11.2021 
 
13 
The study is designed to enrol 220 patients consecutively, where the first 20 patients will partake 
in as a small sub-study at Aarhus University Hospital (study I, the immunological study) and the 
remaining 200 patients will be participating in a larger follow-up study (study II, the survival 
study). 
 
In both studies I and II, CRLM patients will be randomized into two arms: 
• The experimental arm: Patients will be randomized to RFA in addition to liver surgery for 
CRLM in study I and II (Figure 2).  
• In study I, percutaneous RFA will be performed in general anaesthesia one week prior to 
surgery. Blood samples for immune cell analyses as well as translational analyses will be 
collected prior to RFA treatment and again prior to liver surgery in study I, and then post-
operatively on day 1, day 7, and day 28. Surgical biopsies of CRLM and normal tissue will 
be collected peri-operatively. 
• In study II, RFA will be performed peri-operatively 30 minutes before liver surgery. Tissue 
samples from the CRLM and normal liver tissue will be collected peri-operatively, after 
RFA treatment. Blood samples will be collected before the combined RFA and liver sur-
gery begins, and then post-operatively at day 1, day 7, and 28. 
• The control arm: Patients will, as standard care, be randomized to liver surgery for CRLM. 
Tissue samples from the CRLM will collected peri-operatively. Blood samples for immune 
cell analyses and translational analyses will be collected prior to surgery and post-opera-
tively at day 1, day 7, and 28. 
 
                   
 
Figure 2. Randomization for study I and study II in ISCOLIM trial. 
Study I
Study II
Experimental arm
RFA
Surge ry
Surge ry
Surge ry
Surge ry
n:100
n: 10
Baseline blood sample
RFA treatment
Day 0
RFA
Baseline blood sample
CRLM surgery
Peri-operative RFA treatment
Day 1
Day 14
Day 28
CRLM surgery
Day -7
Liver biopsy
Liver biopsy
Liver biopsy

Protocol 
Version 4 
01.11.2021 
 
14 
All 220 included patients will receive the same follow-up 1 month after surgery (Figure 3): Blood 
samples will be collected every 3 months until 3 years of follow-up is concluded; previous studies 
have shown that most patients with recurrence will have tested positive for ctDNA before 18 
months(12, 13). Hereafter, blood samples will be collected each year concomitantly with CT scans. 
Patients will be followed until 5 years of follow-up is concluded. The first interim molecular and 
immunological analyses will be performed when ten patients have been included in each arm, 
(expectantly at 6 months) and then after 12 and 24 months. Concurrently, all included patients will 
undergo standard radiological follow-up with CT scans at 3, 6, 12, 18, 24, 36, 48, and 60 months. 
Translational analyses are explorative, and only CEA results will be available to the treating clini-
cians, unless interim analyses clearly find that translational analyses will improve patient treatment 
 
 
 
Figure 3. Timeline for follow-up program with CT scans and blood samples. 
 
3.2 Study timetable 
Start inclusion: September 2021 
End inclusion: February 2022 
End data collection and follow-up: February 2027 
Interim analyses: 1) After 6 months, 2) September 2022, and 3) September 2023 
Primary endpoint reached, ready to publish: July 2027  
Follow-up: 5 years from last patient included 
 
*Concurrent CT scan
*3 Months
*6 Months
*12 Months
*18 Months
*24 Months
*36 Months
*9 Months
Liver resection
Time line
Blood samples
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
15 Months
21 Months
27 Months
30 Months
33 Months
1 Month
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
8 x 10 ml EDTA
*48 Months
*60 Months

Protocol 
Version 4 
01.11.2021 
 
15 
3.3 Setting 
ISCOLIM is a clinical prospective RCT, initiated at Aarhus University Hospital. During 2021 to 
2022, 220 patients eligible for the trial will be recruited in the treatment responsible hepato-pan-
creatico-biliary surgical departments of Aarhus University Hospital and Rigshospitalet. 
 
3.4 Sample size 
In assessment of the sample size needed to meet the primary objective, we used a 0.05 significance 
level and a desired power of 80%. Assuming that the 1-year survival rate is 90% in the experi-
mental arm and 75% in the control arm, we need 100 patients in each arm (200 in total). Estimates 
are based on clinical experience, as no literature on this subject has been published yet. To be able 
to deal with a total drop-out of ~10%, we aim to include 220 patients (110 in each arm). For 
potential subsequent analyses of 3-year survival, we need 89 patients in each arm, assuming 3-
year survival rates of 75% and 55% in the experimental and control arm, respectively. Thus, our 
study is sufficiently powered to achieve its primary aim.  
 
3.5 Study feasibility 
We need to enrol 220 patients during a period of 18 months, equalling to 12 patients each month. 
We expect to include 4 patients/month at Aarhus University Hospital and 8 patients/month at 
Rigshospitalet. Under normal circumstances, our institutions perform approximately 10 and 30 
operations for CRLM each month, respectively. Thus, we need to include between 30-50% of the 
patients treated at our institutions. From previous research in the departments, we have already 
established the infrastructure and experience with inclusion of patients in clinical trials, and we 
consider the above inclusion rate realistic. The blood and tissue samples will be handled in co-
operation with Regionernes Bio- og Genom bank (RBGB). The collaboration with RBGB is well 
established at both clinical departments. 
 
3.6 Inclusion and exclusion criteria for patients 
All patients with resectable CRLM, planned for liver surgery, will be eligible for inclusion. Prior 
to inclusion, tumor volume will be estimated by CT-volumetry. We will also assess whether the 
CRLM are synchronous or metachronous. 

Protocol 
Version 4 
01.11.2021 
 
16 
3.6.1 Inclusion criteria 
1. Patients diagnosed with metachronous or synchronous CRLM planned for liver resection 
2. At least one tumor with size ³3 cm 
3. Performance status 0-1 
4. Age ³18 years 
 
3.6.2 Exclusion criteria 
1. Liver cirrhosis 
2. Non-curative extrahepatic metastases  
3. Other cancer within 5 years prior to diagnosis 
4. Earlier RFA treatment 
5. Patients who are unlikely to comply with the protocol, unable to return for subsequent visits 
and/or otherwise considered by the PI to be unlikely to complete the study 
 
3.7 Time of inclusion and allocation procedure 
Before arriving to the outpatient clinic, we will evaluate patient’s possibility to participate in 
the study in relation to the department’s diagnostic MDT conference. Data on age, diagnosis, 
comorbidity, and tumor imaging material, which is necessary for patient treatment, will be 
assessed to evaluate if the patient is eligible for inclusion in the study. Potential study subjects 
will then be given the option to participate. CPR numbers will be registered on all eligible 
patients, also patients who decline to participate in the study. Further whether they were 
asked to participate in the study, and whether the accepted or not will be registered. This will 
be done to ensure the quality of the randomisation process, to control for selection bias, and 
enable future cost-benefit analyses. No additional personal information will be obtained be-
fore patient consent. 
 
3.8 Procedures for patient inclusion and informed consent 
Patients, who meet the inclusion criteria are approached in person at the treating surgical depart-
ment. Here, the patients are given written information and verbal information about the project. 
Patients are encouraged to bring an aide to this session. The oral information is given by trained 

Protocol 
Version 4 
01.11.2021 
 
17 
health care professionals (either a liver surgeon or a project nurse with special training). Oral in-
formation is given in an enclosed room to ensure a private conversation. Further, patients are in-
formed about their juridical rights as participants in a research project. Furthermore, as the project 
involves genomic sequencing, which might disclose genetic variants predisposing to specific dis-
eases, the participants are also offered genetic counselling before making their decision to partic-
ipate.  
After the oral information has been given, the patients are allowed at least 24 hours to consider 
their decision to participate, before they can provide a written informed consent form. Informed 
consent is obtained prior to any study-related procedures. The signed and dated consent forms are 
stored in a locked room at the trial office and are available for audit and inspection at any time. 
The written participant information contains contact information to the person being primary re-
sponsible for the project, and to the persons being responsible at the participating departments. 
The information clearly states that these people can be contacted in case of further questions.  
It also mentions that the participant at any time may withdraw his or her consent, causing imme-
diate destruction of all their individual data collected for the project. The consent withdrawal has 
no influence on their relationship to the department or their current/future treatment.  
The written information also mentions that the study involves a biobank, and that residual biolog-
ical material as well as clinical and sequencing data will be transferred to the Colorectal Cancer 
Research Biobank at Aarhus University Hospital for future research. The research biobank is ap-
proved by the Danish Data Protection Agency (j. no. 1-16-02-27-10). All clinical information will 
be obtained and processed in compliance with The Danish Data Protection Act. 
When the residual biological material is used in future research in will be in accordance with cur-
rent data protection rules. Participants can at any time withdraw their consent to storage of residual 
material for future use.  
Further the patient information states that biological samples and data may be shared with other 
international research collaborators, including companies, in a pseudonymized form (non-person-
alized format) for important research purposes, but only if the research purpose is approved ac-
cording to European ethical guidelines.  
 

Protocol 
Version 4 
01.11.2021 
 
18 
3.9 Patient information and consent 
The patient information and consent form are available as Appendix A. 
The patient information states that when the patient gives consent to participate, he or she also 
accept that the PI, and representative of the PI, have direct access to collect the necessary infor-
mation regarding treatment and clinical outcome from medical records and health registries to 
complete the study. This information is required to conduct the study, and for monitoring and 
quality control of the study. All clinical information will be obtained and processed in compliance 
with The Danish Data Protection Act. 
 
3.10 Randomization 
Randomization will be performed using a concealed web-based randomization service managed 
by Aarhus University (REDcap). Randomization will be stratified for centre and performed in 
blocks of ten. Patients will be randomized into two groups (Figure 1): 
Group 1. Additional RFA therapy with liver surgery for CRLM, where the first 10 patients will be 
included in study I and the subsequent 100 patients will be included in study II (experimental arm). 
Group 2. Standard-of-care liver surgery for CRLM, where the first 10 patients will be included in 
study I and the subsequent 100 patients will be included in study II (control arm). 
 
3.10.1 Study Flow 
Gantt chart 
Day -14 
Baseline 
Day -
7 
Day 0 
Day 1 
Day 14 
Day 28 
Mo 
3/6/9/12 
Mo 
15 
Mo 
18 
Mo 
21 
Mo 
24 
Mo 
27/30/33 
Mo 
36/48/60 
Patient  
inclusion 
  
  
  
  
  
  
  
  
  
  
  
  
  
Informed consent 
  
  
  
  
  
  
  
  
  
  
  
  
  
Registration 
of 
baseline data 
  
  
  
  
  
  
  
  
  
  
  
  
  
Randomization 
  
  
  
  
  
  
  
  
  
  
  
  
  
RFA 
  
Study 
I 
Study 
II 
  
  
  
  
  
  
  
  
  
  
Liver surgery 
  
  
  
  
  
  
  
  
  
  
  
  
  
CT scan 
  
  
  
  
  
  
  
  
  
  
  
  
  
CEA 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood  
sampling 
  
Study 
I 
 Pre-
surgery  
 
 
  
  
  
  
  
  
  
Liver biopsy 
  
Study 
I 
  
  
  
  
  
  
  
  
  
  
  
Interim  
analyses 
  
  
  
  
  
  
 ~ 6 & 12  
months 
  
  
  
 
  
  

Protocol 
Version 4 
01.11.2021 
 
19 
Longitudinal 
data 
collection 
  
  
  
  
  
  
  
  
  
  
  
  
  
Blood sample  
analysis 
  
  
  
  
  
  
  
 
 
  
  
  
  
 
Figure 4. Gantt chart with time-schedule and data collection. Blue: Collection from all partici-
pants, Green: Collection from experimental arm only.  
 
3.10.2 Baseline visit (-14 days from operation) 
• Record basic information on all patients eligible for participation: 
-CPR number and surgical centre 
-Patients fulfilling the inclusion criteria are informed about ISCOLIM and asked for con-
sent to participation: 
-register whether the patient accepted or declined participation 
• The following baseline information is collected on participating patients  
-Whether the patients accepted or declined to receive information about incidental genomic 
findings with potential health consequences 
-WHO performance status 
-Height, weight, and Body Mass Index (BMI) 
-Smoking and alcohol use 
-Synchronous or metachronous CRLM 
-Tumor size and histology 
-Number and size of all metastases present 
-Diagnosis of other cancers within 5 years from inclusion 
-Chronic diseases 
-Relevant medication 
-Imaging information 
• Information on randomization result and treatment plan 
 
3.10.3 Month 3 to 60 
• Collection of clinical data (surgical and oncological intervention, and radiological- and 
biochemical evaluations. Specified in 3.15 Collection of clinical information) 

Protocol 
Version 4 
01.11.2021 
 
20 
• End-of-study CT scan and blood samples 
• Collection of radiological, biochemical, and pathological reports on CRLM retrieved for 
use in the statistical analyses 
• Recurrence or residual disease recording 
 
3.11 Radio-frequency ablation (RFA) 
Under guidance of ultrasonography, a single-electrode RFA-needle is placed in the target CRLM, 
with a diameter of a least 3 cm, which is planned for subsequent liver surgery. A coagulation 
necrosis of ~2 cm is created. The RFA needle is removed under the creation of a tract necrosis to 
avoid tumor seeding (tract ablation). At our departments, we have extensive experience with RFA-
treatment of the liver, performing ~150 procedures each year. In the literature, complication rates 
following stand-alone RFA-treatment are low, making it a very feasible procedure(26). The RFA-
treatment will be performed with the patient in general anaesthesia. 
 
3.12 Collection, handling, and biobanking of biological samples 
The biological material is collected and stored in the clinical biobank, Bio and Genome Bank 
Denmark (Danish Cancer Biobank). When this study needs the samples for analysis etc. the project 
will transfer the samples to the “Colorectal Cancer biobank MOMA, AUH”. At the end of the 
study, left-over biological materials, clinical data, and sequencing data will be transferred to the 
“Colorectal Cancer biobank MOMA, AUH” with the aim to facilitate future research projects. 
Patients may at any time request to have his or her samples and data removed from the clinical 
biobanks or destroyed. New projects based on the biobank material will only be initiated after the 
pertinent permissions have been obtained from the relevant authorities and will be conducted in 
compliance with the Danish Data Protection Act. At each blood draw, a total of 80 mL blood is 
collected in eight 10 ml Potassium ethylenediaminetetraacetic acid (EDTA) tubes. For each patient 
in the control arm, 80 ml of blood will be sampled 19 times. For patients in the experimental arm, 
blood will be collected 19 times (study II) or 20 times (study I) (Figure 2 and 3). The blood sam-
ples will be collected pre- and postoperatively to get a baseline biomarker status and then repeat-
edly every three months until 36 months. The last two years of follow-up, blood samples will be 
collected concomitantly with control CT scans. From the samples, CEA are measured, and plasma 

Protocol 
Version 4 
01.11.2021 
 
21 
and buffy coat (nucleated blood cells) are extracted. Drawing, handling, and storage of the 
plasma/buffy coat for analyses is done in accordance with a standard operating procedure (SOP).  
From the isolated plasma, cfDNA will be extracted, analysed for presence of tumor associated 
epigenetic and genetic changes. From the white blood cells of the buffy coat DNA and RNA will 
be extracted and analysed for T-cell receptor changes (see 3.14.). The buffy coat DNA will fur-
thermore be used as a normal reference (patient specific normal DNA) in our efforts to determine 
the tumor specific DNA changes, and methylation patterns in the ctDNA. We will also use the 
buffy coat cells to monitor the tumor associated immune cell changes during the disease course. 
The cells will be used for immune cell characterization with flow cytometry and immune-histo-
chemical assays (see 3.14). DNA and RNA will be extracted from the tumor and normal liver 
biopsies and used to determine tumor specific mutation- and methylome-profiles. The tissue biop-
sies will also be used to examine the immune cell infiltration in the CRLM at a histological level. 
 
3.13 Research biobank and biobank for future research  
When the study is completed, left-over biological materials, clinical data, and sequencing data will 
be transferred to the “Colorectal Cancer biobank MOMA, AUH” with the aim to facilitate future 
research projects. The “Colorectal Cancer biobank MOMA, AUH” has been approved by the Dan-
ish Data Protection Agency (jrl n.1-16-02-27-10). The biological materials, clinical and sequenc-
ing data will remain in the biobank, until the approval expires (currently 1 March 2030) or until 
the patients request their tissue and/or data removed. Patients may at any time request to have his 
or her samples and data removed from the “Colorectal Cancer biobank MOMA, AUH” and de-
stroyed. The storage of tissue and data in the research biobank is done in accordance with the 
Danish Health Act and the General Data Protection Regulation. New projects based on the biobank 
will only be initiated after the pertinent permissions have been obtained from the relevant regula-
tory committees and will abide current law for data protection. Patients will only be included in 
the study if they consent in writing to collection and biobanking of their blood and clinical data. 
 
3.14 Molecular analyses 
3.14.1 CEA analysis 
CEA will be measured in all blood samples by standard laboratory procedure. 

Protocol 
Version 4 
01.11.2021 
 
22 
 
3.14.2 Immunological analyses 
Immunological analyses in study I will be performed on peripheral blood mononuclear cells 
(PBMCs) isolated from the blood sample’s buffy coat and tissue biopsies in a single proliferation 
assay set-up. Flow cytometry will be done using CellTracker labelling with fluorescent monoclo-
nal antibodies on a Quanteon flow cytometer (NovoCyte). Analysis of flow cytometric data will 
be performed by comparing the proliferation as measured by CellTracker labelling for every stim-
ulation after gating for cytotoxic effector T lymphocytes (CD3+CD4+CD8+CD44+CD62L-
CCR7-CD45RA-CD45RO-CD95+ cells), for activated and degranulated cytotoxic T-cells 
(CD3+CD4-CD8+CD62L-CCR7-CD107a+), for central memory effector T cells (CD3+CD4-
CD8+CD27-CD45RA-CCR7+), and for antigen experienced effector memory T cells (CD3+CD4-
CD8+CCR7-CD45RA+CD95+), respectively. In addition, a functional cytotoxic test is under-
taken on fractions of suspended tumor and normal cells. Flow cytometric evaluation of the lymho-
cytotoxic mediated cell death is made by staining tumor cells with (PRF1, CRC_marker(s)) and 
normal cells with (PRF1, hepatocyte_marker(s)). 
 
3.14.3 ctDNA analyses 
ctDNA will be isolated from the longitudinally collected plasma samples using standard methods. 
The presence of ctDNA in the patient’s plasma samples will be used to identify residual disease, 
monitor treatment response and for studies of tumor evolution. The following approaches for de-
tection and quantification of ctDNA will be used: 
1. The TriMeth test: a digital PCR test for detection of circulating DNA fragments with CRC 
specific methylation(27, 28)}. In house, but yet unpublished data, indicate that approxi-
mately 98% of all CRLM patients are TriMeth positive pre-operatively. Postoperatively, 
65% of patients are TriMeth positive, and their 1-year recurrence-free survival is 12%, 
compared to 60% for TriMeth negative patients (P<0.001). 
 
2. Targeted DNA sequencing of cell free plasma DNA to investigate if the plasma contains 
DNA fragments of tumor origin. As marker of the tumor DNA we use tumor specific mu-
tations, copy number changes, and DNA fragmentation patterns. From the sequencing data 
we will quantify the fraction of ctDNA in each sample and correlate this level to treatment 

Protocol 
Version 4 
01.11.2021 
 
23 
response and clinical outcome. We will also explore if the sequencing data can be used to 
assess to tumor evolution during treatment and surveillance. Illumina TruSeq DNA Kit and 
NimbleGen SeqCap EZ v3.0 (or similar) will be used for generation of sequencing libraries 
and capture. Data will be generated on the Illumina NovaSeq platform. The target sequence 
coverage is >1,000x. 
 
3.14.4 T-cell receptor analyses 
T-cell Receptor sequencing (TCR-Seq) will be performed to identify the role of the adaptive im-
mune system stimulated by RFA exposure in the ISCOLIM trial. We will do TCR-Seq on DNA 
and RNA extracted from tumor tissue and from the longitudinally collected blood samples to 1) 
determine the TCR profiles in the tissues, 2) establish the TCR profiles of the blood, 3) monitor 
changes in the TCR repertoire over time and in response to treatment, and 4) to compare the pro-
files of blood and tissue. 
For TCR-seq we will use the AmpliSeq for Illumina Immune Repertoire Plus, TCR beta Panel, 
which is a highly multiplexed targeted resequencing panel to measure T cell diversity and clonal 
expansion by sequencing T cell receptor (TCR) beta chain rearrangements. Data will be generated 
on the Illumina NovaSeq platform. Target coverage is >1,000x. 
 
3.14.5 DNA sequencing 
We will apply whole genome sequencing, as well as targeted sequencing of smaller cancer panels 
to tumor and germline DNA to identify tumor specific genomic changes, clonality, and neoanti-
gens. Illumina TruSeq DNA Kit and NimbleGen SeqCap EZ v3.0 (or similar) will be used for 
generation of sequencing libraries and capture. Data will be generated on the Illumina NovaSeq 
platform. Target coverage is <100x. 
 
3.14.6 DNA Methylation analyses 
We will extract DNA from the CRLM and normal liver tissue and establish DNA methylation 
profiles of the two tissues by analysing the DNA with the Infinium Methylation EPIC Array, 850K 
CpG sites (or a similar approach). We will explore if specific methylation patterns are associated 

Protocol 
Version 4 
01.11.2021 
 
24 
with response to ISCOLIM treatment. We will also explore if the immune cell compositions can 
be inferred from the DNA methylation profiles.  
 
3.14.7 RNA sequencing  
Total RNA-seq will be used for identification of composition and gene expression of tumor-infil-
trating immune cells associated with treatment and tumor evolution. Further, RNA-seq will be 
used to investigate T-cell receptor clonality. The ScriptSeq protocol (or similar) will be used for 
generation of libraries. Data will be generated on the Illumina NovaSeq platform. As coverage 
varies with expression level, there is no target coverage. However, we aim to generate 100 mio 
sequencing reads per sample. 
 
3.14.8 Bioinformatics and Data analyses 
Multi-dimensional omics data from ctDNA, DNA, and RNA analyses on tumor and blood will be 
analysed and integrated in collective, predictive models for treatment response and survival (re-
currence free-, disease free- and overall survival). Specifically, we will process the DNA and RNA 
sequencing data through MOMA’s well-established analytic pipelines. For DNA, data will be 
aligned and calibrated by picard and GATK suites. SNPs will be identified by GATK Haplotype-
Caller, and somatic mutations (SNVs) and indels will be identified with Mutect2 and Strelka2 (or 
similar software). 
 
3.15 Collection of clinical information 
To enable recruitment, patient’s age, diagnosis, comorbidity, and tumor imaging material will be 
evaluated on all patients eligible for participation. Specifically, information about surgical inter-
vention, pathology reports, oncological intervention, and radiological evaluations aiming at de-
tecting disease recurrence or assessing changes in tumor burden (e.g. during surveillance and treat-
ment) will be collected. Data will be collected for up to ten years after the last protocolled patient 
visit, and is required to conduct the study, to use in the statistical analyses, and for monitoring and 
quality control of the study. 

Protocol 
Version 4 
01.11.2021 
 
25 
For participating patients, the following information will be obtained from medical records; CPR 
number, surgical centre, age, gender, height, weight, BMI, WHO performance status, smoking/al-
cohol, and relevant medication use. Further, tumor size and histology, synchronous or metachro-
nous CRLM, number of metastases, diagnosis of other cancers within 5 years, and chronic diseases 
will be recorded. Patients who consent to participate, further accept that a central element of the 
project involves comparison, correlation, and assessment of ctDNA and immune responses (gath-
ered from the blood samples) to cancer treatment and clinical outcome. All clinical information 
will be obtained and processed in compliance with data protection laws. 
3.16 Definitions of predictor variables, co-variates, and endpoints 
3.16.1 Primary endpoint  
• Fraction of patients with recurrence receiving RFA and curative resection, or curative re-
section only. 
• Overall survival at 1, 3, and 5 years. 
 
3.16.2 Secondary endpoints  
• DFS 
• RFS 
• Time to clinical recurrence (TTCR) 
• Time to molecular recurrence (TTMR) by ctDNA, TCR, or CEA measures 
• Adherence rate   
 
3.16.3 Safety Endpoints 
• Frequency and severity of adverse events (AE) in relation to the per protocol blood draws. 
• Allergic reaction to contrast material in relation to CT imaging. 
• Complications in relation to RFA treatment (bleeding, hepatic abscesses, hepatic infarcts, 
and bile duct injuries). 
• Complications in relation to general anaesthesia twice in two weeks (Study I). 
 

Protocol 
Version 4 
01.11.2021 
 
26 
3.16.4 Definition of endpoints 
• Overall survival is defined as the time from surgery to death from any cause.  
• Time to clinical recurrence is calculated from date of surgery until loco-regional recurrence 
or distant metastases, or sudden death from CRLM.  
• Time to molecular recurrence is calculated from first time of no detectable ctDNA until 
detectable ctDNA. 
• Adherence rate is defined as the proportion of patients adhering 100% to the protocol.  
 
3.17 Statistical analyses 
3.17.1 Demographics and baseline data  
All data will be presented using descriptive statistics. Continuous variables will be summarized 
using mean, standard deviation, median, minimum, and maximum. Categorical variables will be 
summarized using the number and percentage of patients. 
 
3.17.2 Analysis of OS, TTCR, and TTMR 
Data will be analyzed as intention-to-treat. Kaplan-Meier estimates will be used for the estimation 
of median times to clinical recurrence, disease or death, and their confidence intervals stratified 
according to follow-up intensity. Endpoints will be assessed using the log-rank test or a Cox re-
gression model, with time to event (clinical or molecular recurrence or death) as response variable 
and RFA as a factor. The difference in clinical recurrence versus molecular recurrence will be 
compared using paired t-tests and regression analyses.  
We will follow the patients from the date of surgery until disease recurrence, death, or censoring 
within the five years follow-up. Patients will undergo standard postoperative surveillance CT scans 
every 3 months the first year and every 6 months hereafter until 36 months (Fig.3). Beyond the 
third year, CT scans will be performed annually. 
To avoid unintentional bias, the demographics of patients not willing to participate in the trial will 
be compared to the study population. Statistics will be performed in close collaboration with pro-
fessional health research statisticians. 
 

Protocol 
Version 4 
01.11.2021 
 
27 
3.17.3 Analysis of Safety 
AEs will be reported to the PI. All AEs will be coded according to the standardized Medical Dic-
tionary for Regulatory Activities (MedDRA). The coding will be performed by a researcher ap-
pointed by the PI. AEs will be summarized by presenting their incidence of AEs, based on the 
numbers and percentages of patients with AEs. 
 
4. Data management 
Data from the study will be collected from medical records and from the Danish Colo-rectal Cancer 
Group database (DCCG database). The information from the medical records will be entered into 
the study database, e.g. baseline information, pathology data, date of visits, and radiological con-
trol visits. Other information will be imported to the study database from the DCCG database e.g. 
tumor characteristics. Other external data e.g. laboratory data will be entered or transferred into 
the study database. 
 
4.1 Quality Control 
A quality control (QC) of data will be performed to ensure that data entry and verification have 
been performed correctly in accordance with pre-defined instructions. The QC will be performed 
before data is declared clean. 
The trial sites will be visited by the Clinical Trial Manager (Monitor) appointed by the PI. The 
Monitor will visit periodically at times agreed with the PI. It is the function of the Monitor to 
ascertain that all aspects of the protocol are complied with and that the conduct of the trial con-
forms to applicable regulatory requirements and established rules for Good Clinical Practice 
(GCP). Preferably at the time of each monitoring visit, the Monitor will review the collected data 
to ascertain that items have been completed and that the data provided are accurate and obtained 
in the manner specified in the protocol. Further, the Monitor will verify that the data from external 
databases are consistent with the clinical records or other relevant records (Source Data Verifica-
tion) and that trial results are recorded completely and correctly. For this purpose, the Monitor 
must be given direct access to clinical records, original laboratory data, etc., as far as these relate 
to the trial and without jeopardizing patient integrity. The investigators and other relevant person-
nel should be available during the monitoring visits and should devote sufficient time. 

Protocol 
Version 4 
01.11.2021 
 
28 
All investigators and staff carrying out observations of primary or other major efficacy variables 
involved in the trial should provide a curriculum vitae. The PI will keep a list of all personnel 
involved in the trial together with their function and trial-related delegated duties. He will ensure 
that appropriate trial-related training is given to all staff, and that any new information of relevance 
to the performance of this trial is forwarded to the staff involved. Before inclusion of patients, the 
Monitor will perform a trial initiation visit to inform and train relevant trial staff.  
Any substantial change to the approved Final Trial Protocol will be documented in a written and 
numbered Protocol Amendment. Any proposed substantial change to the Final Trial Protocol must 
be discussed with and approved by the Sponsor before submitted to the relevant Regulatory Au-
thority for approval. The study will be registered at ClinicalTrials.gov and monitored by the GCP 
Unit at Aarhus University Hospital. 
 
 
 

Protocol 
Version 4 
01.11.2021 
 
29 
5. Processing of personal data in the ISCOLIM trial study 
This project is registered at the internal list of research projects in the Central Denmark Region. 
We will obtain approval from the local scientific ethics committee prior to inclusion of the first 
patient. We will adhere to the Helsinki declaration, and all data will be handled in accordance with 
The Danish Data Protection Act (“Databeskyttelsesforordningen” and “Databeskyttelsesloven”). 
Before study initiation processing of the patient’s personal data will be registered in the appropriate 
system at the Central Denmark Region. Further, the trial will be approved by the Central Denmark 
Region Ethics Committee. No patient will be included before all permissions are granted. The 
study is covered by the Danish Patient Compensation Authority.  
Throughout the study, all clinical data and samples will be labelled with non-personal identifiers. 
Information to identify individual patients will only be available to the PI (Prof. Frank Viborg 
Mortensen) and the TI (Prof. Claus Lindbergh Andersen) and will only be used when collecting 
clinical information from the patient. The molecular and clinical data produced in the study will 
be stored on keyword protected and log-file operated servers operated by Aarhus University and 
Aarhus University Hospital. In accordance with good academic practice and the requirements of 
the bodies funding the study and the scientific journals publishing the study results, the study data 
(health data and genomic data), in pseudonymized format, will be transferred to the secure data-
base ”European Genome-Phenome Archive, (EGA)” (https://ega-archive.org). This will happen 
after the study has been completed. The purposes are to enable sharing of the data with other 
research groups, inside and outside Denmark, confirmation of the study findings as well as future 
research. The EGA provides the necessary security required to control access, and maintain patient 
confidentiality, while providing access to those researchers and clinicians authorised to view the 
data. In all cases, data access decisions will be made by the ISCOLIM steering committee and not 
by the EGA. Data sharing will be conducted in accordance with the European data protection reg-
ulations, including The Danish Data Protection Act and the General Data Protection Regulation 
(GDPR). Before making a data access decision, the ISCOLIM steering committee will consult the 
Central Denmark Region Legal Office and data sharing will only take place after all necessary 
approvals have been obtained from the relevant Danish authorities. 
The EGA is part of the European ELIXIR research infrastructure. ELIXIR is an intergovernmental 
organization that unites Europe’s leading life science organisations in managing and safeguarding 

Protocol 
Version 4 
01.11.2021 
 
30 
the increasing volume of data being generated by publicly funded research. It coordinates, inte-
grates and sustains bioinformatics resources across its member states and enables users in aca-
demia and industry to access services that are vital for their research. These resources include 
databases, software tools, training materials, cloud storage and supercomputers (https://elixir-eu-
rope.org/about-us). ELIXIR is partly funded by the European Commission within the Research 
Infrastructures programme of Horizon 2020. Denmark is an ELIXIR node. An ELIXIR Node is a 
collection of research institutes within a member country. ELIXIR nodes run the resources and 
services that are part of ELIXIR. The EGA is hosted by the ELIXIR Nodes of Spain and United 
Kingdom. Within the EU it is the Spanish ELIXIR node that runs the EGA, specifically it is the 
Centre for Genomic Regulation (CRG), C/ Dr. Aiguader 88, PRBB Building, 08003 Barcelona, 
Spain. CRG is a non-profit foundation funded by the Catalan Government through the Department 
of Business & Knowledge and the Department of Health, the Spanish Ministry of Science & In-
novation, the "la Caixa" Banking Foundation, and includes the participation of Pompeu Fabra Uni-
versity. 
 
 
6. Ethics and risks 
6.1. RFA 
The RFA-treatment used in this study is a minimally invasive procedure, with a low risk of com-
plications. Thus, it is feasible in most patients. Potential RFA-related risks are bleeding and infec-
tion (1-2%). Extremely rare complications are hepatic abscesses, hepatic infarcts, and bile duct 
injuries. In ten patients, RFA will be performed separately from liver surgery (Study I). Follow-
ingly, these patients will undergo general anaesthesia twice. However, general anaesthesia in elec-
tive surgery is a very safe procedure with limited risks. The most common side effect is nausea 
and vomiting, which will be treated with antiemetics. As the potential clinical impact of this study 
is a possible increased long-term survival due to the reduced risk of cancer recurrence, the benefits 
of participating in the experimental arm outweigh the few potential side effects. 
 

Protocol 
Version 4 
01.11.2021 
 
31 
6.2. Blood samples 
Clinical blood samples will be drawn 19 times for patients in the control arm and likewise for 
patients in the experimental study II. In study I, blood samples will be drawn 20 times. Per draw, 
80 ml of blood will be collected. The blood samples will be used to measure CEA, a biomarker 
that guides clinical treatment decisions, as well as explorative translational biomarkers, that could 
improve onco-surgical treatment of CRLM patients.  
The lost blood volume, contributed to each blood draw, is clinically irrelevant (<2% of the total 
blood volume) for the patient’s health, especially in patients with performance status 0-1. Blood 
sampling is associated with minimal risk and discomfort to the patients, and rarely causes side-
effects (hematoma, rash, or infection). To limit the inconvenience as much as possible, the blood 
samples will be drawn as part of the routine management, whenever this is possible. 
 
6.3 Tissue samples 
Tissue samples from CRLM and normal liver tissue are collected from the resected part of the 
liver, as needle biopsies and as surgical biopsies. The needle biopsies will be collected peri-oper-
atively with ultrasound guidance concurrently with the RFA treatment before surgery and does not 
add to the overall procedural risk.  
 
Liver biopsy is a safe procedure that causes few adverse effects and can be performed in an out-
patient setting. Risks includes infection and bleeding (1-2%). To avoid the risk of tumor seeding, 
needle biopsies are taken through a sheet. Both needle biopsies and surgical biopsies are taken 
peri-operatively and does not affect the patient or the pathological evaluation of the CRLM. 
 
6.4 CT scans 
The patients included in ISCOLIM will undergo standard post-operative surveillance CT scans. 
No additional CT scans will be performed due to this study and thus, there is no additional radiation 
risk in participating in the study. 
 

Protocol 
Version 4 
01.11.2021 
 
32 
6.5 Genomic sequence analysis 
In this study, we will DNA and RNA sequencing primarily to determine: 1) the TCR repertoire, 
2) establish the somatic tumor mutation profile of each tumor, 3) detect and quantify ctDNA, and 
4) establish gene expression profiles. We will not search the data for germline genetic variation 
(e.g. SNPs) s. Consequently, the risk of incidental finding a potential clinically relevant, genomic 
variant related to inherited diseases is extremely low, and practically hypothetical. Nevertheless, 
in the unlikely event that we do identify a genetic variant with potential clinical relevance, then 
we will have its importance evaluated by an expert committee, appointed by MOMA. 
The committee is appointed when needed and the members will be chosen according to the poten-
tial disease. The committee will include a molecular biologist, specialized in genetic sequencing, 
a medical doctor specialized in personalized medicine, a clinical geneticist specialized in inherited 
diseases, and a medical doctor specialized in the disease in question. If deemed relevant other 
specialists may be included. 
This committee will assess if  
1. The technological quality of the analysis is sufficient for a reliable result. 
2. There is sufficient evidence in the literature for a clinical relevance (e.g. expected pene-
trance). 
3. The sum of information justifies a relevant risk for a genetic disposition. 
4. The disease, according to current standards, can be treated or prevented.  
Based on the assessments, the committee decides whether the patient should be informed (by writ-
ten letter) that the research accidentally has resulted in a finding, with potential influence on his or 
her health. The patient will also be informed that further information and advice on the matter will 
be offered to him/her and/or potentially affected family members.   
If the patient in the consent form opted out of receiving important health-related findings or has 
died, the committee will assess whether to contact relatives, with purpose of saving lives and pre-
venting disability.  
 
 
 

Protocol 
Version 4 
01.11.2021 
 
33 
7. Publication and reporting from the trial 
The results of the ISCOLIM trial are expected to be published in international scientific journals. 
The reporting will follow the CONSORT guidelines for reporting randomized controlled trials 
(http://www.consort-statement.org/). Positive, negative, and non-definable findings will be pub-
lished and presented at international scientific conferences as soon as at it is considered profes-
sionally sound. We will aim for publications in high-impact journals with focus on oncology, ra-
diology, and liver surgery. If results unexpectedly cannot be published in journals, other methods 
of publication will be sought, such as the collaborators’ institutional websites. 
 
8. Economy 
This study is sponsor-initiated. The study is supported by two research grants given to prof. Frank 
Viborg Mortensen (Novo Nordisk Fonden [2.982.000 DKK] and Neye Fonden [675.000 DKK]). 
Grants from these foundations are administered by Aarhus University Hospital. The grants support 
running costs related to the project (blood samples, RFA needles, project nurses etc.). The inves-
tigators and collaborators have no financial relation to the grant providers, nor any financial inter-
est in the trial. Salaries to the research staff are already financed by their respective employers. 
 
8.1 Remuneration to participants 
There is no remuneration to the study participants. 
 
8.2 Patient compensation scheme (patienterstatningen) 
The study is covered by the Danish Patient Compensation Scheme (patienterstatningsordningen). 
 
9. References 
1. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pineros M, et al. Estimating 
the global cancer incidence and mortality in 2018: Globocan sources and methods. Int J 
Cancer 2019;144:1941-53. 
2. database Nc. Nordcan. Vol.  https://www-dep.iarc.fr/NORDCAN/DK/frame.asp: IACR, CSU, 
2018. 

Protocol 
Version 4 
01.11.2021 
 
34 
3. Hackl C, Neumann P, Gerken M, Loss M, Klinkhammer-Schalke M, Schlitt HJ. Treatment of 
colorectal liver metastases in germany: A ten-year population-based analysis of 5772 cases 
of primary colorectal adenocarcinoma. BMC Cancer 2014;14:810. 
4. Boysen AK, Spindler KL, Hoyer M, Mortensen FV, Christensen TD, Farkas DK, Ording AG. 
Metastasis directed therapy for liver and lung metastases from colorectal cancer-a 
population-based study. Int J Cancer 2018;143:3218-26. 
5. Chakedis J, Schmidt CR. Surgical treatment of metastatic colorectal cancer. Surg Oncol Clin N 
Am 2018;27:377-99. 
6. Engstrand J, Nilsson H, Stromberg C, Jonas E, Freedman J. Colorectal cancer liver metastases 
- a population-based study on incidence, management and survival. BMC Cancer 
2018;18:78. 
7. Kanas GP, Taylor A, Primrose JN, Langeberg WJ, Kelsh MA, Mowat FS, et al. Survival after 
liver resection in metastatic colorectal cancer: Review and meta-analysis of prognostic 
factors. Clin Epidemiol 2012;4:283-301. 
8. Manfredi S, Lepage C, Hatem C, Coatmeur O, Faivre J, Bouvier AM. Epidemiology and 
management of liver metastases from colorectal cancer. Ann Surg 2006;244:254-9. 
9. Bedin C, Enzo MV, Del Bianco P, Pucciarelli S, Nitti D, Agostini M. Diagnostic and prognostic 
role of cell-free DNA testing for colorectal cancer patients. Int J Cancer 2017;140:1888-
98. 
10. Spindler KG, Boysen AK, Pallisgard N, Johansen JS, Tabernero J, Sorensen MM, et al. Cell-
free DNA in metastatic colorectal cancer: A systematic review and meta-analysis. 
Oncologist 2017;22:1049-55. 
11. Basnet S, Zhang ZY, Liao WQ, Li SH, Li PS, Ge HY. The prognostic value of circulating cell-
free DNA in colorectal cancer: A meta-analysis. J Cancer 2016;7:1105-13. 
12. Reinert T, Scholer LV, Thomsen R, Tobiasen H, Vang S, Nordentoft I, et al. Analysis of 
circulating tumour DNA to monitor disease burden following colorectal cancer surgery. 
Gut 2016;65:625-34. 
13. Scholer LV, Reinert T, Orntoft MW, Kassentoft CG, Arnadottir SS, Vang S, et al. Clinical 
implications of monitoring circulating tumor DNA in patients with colorectal cancer. Clin 
Cancer Res 2017;23:5437-45. 

Protocol 
Version 4 
01.11.2021 
 
35 
14. Tie J, Wang Y, Tomasetti C, Li L, Springer S, Kinde I, et al. Circulating tumor DNA analysis 
detects minimal residual disease and predicts recurrence in patients with stage ii colon 
cancer. Sci Transl Med 2016;8:346ra92. 
15. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of 
circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 
2014;6:224ra24. 
16. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA 
from maternal plasma. Am J Hum Genet 1999;64:218-24. 
17. Tie J, Cohen JD, Wang Y, Li L, Christie M, Simons K, et al. Serial circulating tumour DNA 
analysis during multimodality treatment of locally advanced rectal cancer: A prospective 
biomarker study. Gut 2019;68:663-71. 
18. Solbiati L, Ahmed M, Cova L, Ierace T, Brioschi M, Goldberg SN. Small liver colorectal 
metastases treated with percutaneous radiofrequency ablation: Local response rate and 
long-term survival with up to 10-year follow-up. Radiology 2012;265:958-68. 
19. Crocetti L, de Baere T, Lencioni R. Quality improvement guidelines for radiofrequency 
ablation of liver tumours. Cardiovasc Intervent Radiol 2010;33:11-7. 
20. Evrard S, Poston G, Kissmeyer-Nielsen P, Diallo A, Desolneux G, Brouste V, et al. Combined 
ablation and resection (care) as an effective parenchymal sparing treatment for extensive 
colorectal liver metastases. PLoS One 2014;9:e114404. 
21. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell 2011;144:646-74. 
22. Pages F, Galon J, Dieu-Nosjean MC, Tartour E, Sautes-Fridman C, Fridman WH. Immune 
infiltration in human tumors: A prognostic factor that should not be ignored. Oncogene 
2010;29:1093-102. 
23. Varade J, Magadan S, Gonzalez-Fernandez A. Human immunology and immunotherapy: Main 
achievements and challenges. Cell Mol Immunol 2020. 
24. Filin IY, Solovyeva VV, Kitaeva KV, Rutland CS, Rizvanov AA. Current trends in cancer 
immunotherapy. Biomedicines 2020;8. 
25. den Brok MH, Sutmuller RP, van der Voort R, Bennink EJ, Figdor CG, Ruers TJ, Adema GJ. 
In situ tumor ablation creates an antigen source for the generation of antitumor immunity. 
Cancer Res 2004;64:4024-9. 

Protocol 
Version 4 
01.11.2021 
 
36 
26. Fonseca AZ, Santin S, Gomes LG, Waisberg J, Ribeiro MA, Jr. Complications of 
radiofrequency ablation of hepatic tumors: Frequency and risk factors. World J Hepatol 
2014;6:107-13. 
27. Jensen SO, Ogaard N, Nielsen HJ, Bramsen JB, Andersen CL. Enhanced performance of DNA 
methylation markers by simultaneous measurement of sense and antisense DNA strands 
after cytosine conversion. Clin Chem 2020;66:925-33. 
28. Jensen SO, Ogaard N, Orntoft MW, Rasmussen MH, Bramsen JB, Kristensen H, et al. Novel 
DNA methylation biomarkers show high sensitivity and specificity for blood-based 
detection of colorectal cancer-a clinical biomarker discovery and validation study. Clin 
Epigenetics 2019;11:158.