Implementing Non-invasive Circulating Tumor DNA Analysis to Optimize the Operative and Postoperative Treatment for Patients With Colorectal Cancer - Intervention Trial 2
Phase: NA
Conditions: Colorectal Cancer, Colo-rectal Cancer, ctDNA, Gastro-Intestinal Disorder, Colorectal Neoplasms, Gastrointestinal Cancer, Gastrointestinal Neoplasms, Digestive System Disease, Digestive System Neoplasm, Colonic Diseases, Colonic Neoplasms, Colonic Cancer, Rectal Diseases, Rectal Neoplasms, Rectal Cancer
Sponsor: Claus Lindbjerg Andersen

IMPROVE-IT2: Implementing non-invasive circulating tumor DNA analysis to optimize the operative and 
postoperative treatment for patients with colorectal cancer – Intervention Trial 2 
NCT04084249 
June 21st, 2024 
 
Ethical approvals and amendments 
Type 
Changes – brief description 
Date 
Status 
Main protocol 
- 
27-08-2019 
Approved 
Amendment 1 
Adjustments of protocol to comply with SPIRIT 
guidelines 
06-09-2019 
Approved 
Amendment 2 
Adjustments of the following: 
- 
Inclusion criteria 
- 
Timing of QoL questionnaire,  
- 
Timing of patient inclusion 
- 
Randomization strategy 
- 
Evaluation of primary objective 
Addition of the following: 
- 
 “European Quality of Life” questionnaire 
- 
Description of the targeted quality of PET/CT 
scans 
- 
A second opinion radiology committee 
- 
Description of quality of life and health 
economy analyses 
02-12-2019 
Approved 
Amendment 3 
Clariﬁcation of how approached patients that decide 
not to participate, are registered in the trial database 
for end-of-trial demographic comparison of the 
approached and the enrolled patient populations. 
10-01-2020 
Approved 
Amendment 4 
Extension of the study recruitment period due to 
COVID-19 
06-11-2020 
Approved 
Amendment 5 
Addition of a secondary objective regarding ctDNA 
growth rates, and 2 extra blood samples for ctDNA-
positive patients in the experimental arm. 
Increased sample size (n = 310) due to newly published 
data showing that contemporary recurrence rates of 
high-risk stage II and stage III patients were lower than 
expected when the sample-size calculation was initially 
conducted. 
13-06-2022 
Approved 
Amendment 6 
Adjustments of protocol procedures regarding the 36-
month blood sample. 
06-08-2022 
Approved 
Amendment 7 
Increased the needed sample size to n = 340, due to a 
competing study leading to a larger than anticipated 
drop-out rate. 
29-11-2022 
Approved 
Amendment 8 
Addition of ﬁnger-prick blood spot sample collection for 
ctDNA-positive patients in the experimental arm. 
29-01-2024 
Approved 
Amendment 9 
Aligning the objectives and endpoints with the 
published statistical analysis plan. 
03-07-2024 
Approved 
 

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IMPROVE-IT2  
Implementing non-invasive circulating tumor DNA 
analysis to optimize the operative and postoperative 
treatment for patients with colorectal cancer – 
Intervention Trial 2 
 
 
 
Primary Investigator, Clinical 
Associate Professor, Specialty Registrar Kåre Andersson Gotschalck 
Department of Surgery 
Regional Hospital, Horsens  
Sundvej 30 
DK8700 Horsens, Denmark 
+45 26497569   
kaarsune@rm.dk 
 
Sponsor and Primary Investigator, 
Molecular Biology 
Professor Claus Lindbjerg Andersen 
Department of Molecular Medicine 
Aarhus University Hospital  
Palle Juul-Jensens Boulevard 99 
DK8200 Aarhus N, Denmark 
+45 7845 5319 
+45 2980 4321 
cla@clin.au.dk 
 
 
 
Trial Office  
IMPROVE trial office 
Department of Molecular Medicine, Aarhus University Hospital,  
Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark  
Phone: +45 7845 5325 
E-mail: demuth@clin.au.dk  
 
 
 
 

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Protocol signature page 
 
Primary Investigators and Sponsor  
 
Trial title: 
IMPROVE-IT2 trial: Implementing non-invasive circulating tumor DNA analysis to optimize the operative and 
postoperative treatment for patients with colorectal cancer – Intervention Trial 2 
 
 
 
 
Primary Investigator, Clinical  
 
Dr Kåre Andersson Gotschalck 
 
Signature 
 
Date 
 
Sponsor and Primary Investigator, Molecular Biology 
 
Professor Claus Lindbjerg Andersen 
 
Signature 
 
Date 
 
 
 

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Investigator signature page 
 
Investigator 
 
Trial title: 
IMPROVE-IT2 trial: Implementing non-invasive circulating tumor DNA analysis to optimize the operative and 
postoperative treatment for patients with colorectal cancer – Intervention Trial 2 
 
“I agree to the terms of this trial protocol. I will conduct the study in accordance with the procedures 
specified in the protocol, the ethical principles in the latest version of the Declaration of Helsinki, Good 
Clinical Practice and applicable regulatory requirements”. 
 
 
 
 
 
 
Center  
 
Investigator name 
 
Signature 
 
Date 
 
 
 

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Trial administrative structure 
This is an investigator-initiated study. Associate Professor, Specialty Registrar Kåre Andersson Gotschalck is 
the clinical coordinating investigator. Professor Claus Lindbjerg Andersen is the sponsor and the coordinator 
of the molecular analyses. The decision-making body of the study is the steering committee (SC), which will 
have representation from all participating departments. Professor Claus Lindbjerg Andersen is chair of the 
SC. The steering committee oversees the progress and funding of the trial, and determines the strategy for 
the execution of the trial. 
It is the role of the sponsor to ensure funding to conduct the trial. 
The organizations and foundations supporting the trial have no influence on design, execution or reporting 
from the trial. 
It is the role of the clinical coordinating investigator and the sponsor to define the trial design and to ensure 
that the trial data is collected, analyzed and published.  It is the role of the clinical coordinating investigator 
to oversee clinical part of the trial and to ensure that all trial personnel gets the necessary training to conduct 
their tasks in the trial. It is the task of the clinical coordinating investigator in collaboration with the 
coordinating center and sponsor solve the problems that occur during trial. 
It is the role of the clinical coordinating investigator and sponsor to ensure that the trial at all times is 
conducted in agreement with the Danish legislation.  
The role of the coordinating center is to ensure establishment of the necessary trial infrastructure, including 
databases, trial guidelines, and trial documents. The coordinating center houses the Clinical Trial Manager 
who’s task it is to monitor the trial sites and the study database, and verify the quality of the data collected.  
It is the task of the Sponsor to ensure that audits and inspections of the trial sites are conducted to assure 
the quality of trial processes, procedures and data.   
Surgical departments in Denmark with a defined high volume of colorectal resections can participate in the 
study and will be invited to do so. 
 
A list of participating study sites can be obtained from the clinical trial manager.  
 
 
 

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Table of contents 
Protocol signature page .................................................................................................................................... 2 
Investigator signature page ............................................................................................................................... 3 
Trial administrative structure ............................................................................................................................ 4 
Table of contents ............................................................................................................................................... 5 
1 
Introduction ............................................................................................................................................... 8 
2 
Objectives ................................................................................................................................................ 10 
2.1 
Primary Objective ............................................................................................................................ 10 
2.2 
Key Secondary Objectives - Oncological .......................................................................................... 10 
2.2.1 
Key Secondary Objective 1 (S1) ............................................................................................... 10 
2.2.2 
Key Secondary Objective (S2) .................................................................................................. 10 
2.3 
Key Secondary Objectives – Quality of Life ..................................................................................... 10 
2.3.1 
Key Secondary Objective 3 (S3) ............................................................................................... 10 
2.4 
Secondary Objectives ...................................................................................................................... 10 
2.4.1 
Secondary Objective 4 (S4) ...................................................................................................... 10 
2.4.2 
Secondary Objective 5 (S5) ...................................................................................................... 10 
2.4.3 
Secondary Objective 6 (S6) ...................................................................................................... 11 
2.4.4 
Secondary Objective 7 (S7) ...................................................................................................... 11 
2.4.5 
Secondary Objective 8 (S8) ...................................................................................................... 11 
2.4.6 
Secondary Objective 9 (S9) ...................................................................................................... 11 
2.4.7 
Secondary Objective 10 (S10) .................................................................................................. 11 
3 
Investigational plan ................................................................................................................................. 11 
3.1 
Overall study design ........................................................................................................................ 11 
3.2 
Study timetable ............................................................................................................................... 12 
3.3 
Setting .............................................................................................................................................. 12 
3.4 
Sample size ...................................................................................................................................... 12 
3.5 
Inclusion of patients ........................................................................................................................ 13 
3.5.1 
IMPROVE-IT2 patient criteria .................................................................................................. 13 
3.5.1.1 
Inclusion criteria .................................................................................................................. 13 
3.5.1.2 
Exclusion criteria .................................................................................................................. 13 
3.5.2 
Time of inclusion and allocation procedure ............................................................................ 14 
3.5.3 
Procedures for patient recruitment and informed consent .................................................... 14 
3.5.4 
Patient information and consent............................................................................................. 15 
3.6 
Randomization ................................................................................................................................. 15 

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3.7 
Quality of Life baseline consideration: ............................................................................................ 15 
3.8 
Study flow ........................................................................................................................................ 16 
Post OP day 14 visit (inclusion visit, Baseline) ......................................................................................... 17 
3.8.1 
Post OP month 3 (Status of adjuvant therapy, EXCLUSION of patients NOT treated with 
adjuvant therapy, RANDOMIZATION of patients treated with adjuvant therapy, send QoL to patients 
expected to end adjuvant therapy before month 4) ............................................................................... 18 
3.8.2 
Post OP month 4 visit (collection of a blood sample, and information about randomization)19 
3.8.3 
Post OP month 7 (Status of adjuvant therapy for all patients who were still in active 
treatment at the month 4 visit) ............................................................................................................... 19 
3.8.4 
Post OP month 8 visit .............................................................................................................. 20 
3.8.5 
Longitudinal post OP visits (mo12 to mo36) ........................................................................... 20 
3.8.5.1 
For patients in the Control arm: .......................................................................................... 20 
3.8.5.2 
For patients in the Experimental arm .................................................................................. 21 
3.8.5.2.1 
Experimental arm, ctDNA negative: .............................................................................. 21 
3.8.5.2.2 
Experimental arm, ctDNA positive: ............................................................................... 21 
3.9 
CT-scan standard ............................................................................................................................. 22 
3.10 
PET-CT-scan technical standard ....................................................................................................... 23 
3.11 
Collection, handling, and biobanking of biological samples............................................................ 23 
3.11.1 
Collection and handling of blood samples............................................................................... 23 
3.11.2 
Research Biobank for the IMPROVE study: ............................................................................. 23 
3.11.3 
Biobank for future research: ................................................................................................... 23 
3.12 
Molecular analysis ........................................................................................................................... 23 
3.12.1 
Detection and quantification of circulating tumor DNA.......................................................... 23 
3.12.1.1 
ctDNA detection by digital PCR ....................................................................................... 24 
3.12.1.2 
ctDNA detection by ultra-deep targeted sequencing ...................................................... 24 
3.12.1.3 
ctDNA detection by low pass whole genome sequencing............................................... 25 
3.12.1.4 
Bioinformatic processing of sequencing data ................................................................. 25 
3.12.1.5 
Storage of sequencing data ............................................................................................. 25 
3.12.2 
Quantification of carcinoembryonic antigen (CEA) ................................................................. 25 
3.13 
Collection of clinical Information .................................................................................................... 25 
3.14 
Definitions of predictor variables, co-variates and end-points ....................................................... 26 
3.14.1 
Primary endpoint ..................................................................................................................... 26 
3.14.2 
Secondary endpoints ............................................................................................................... 26 
3.14.3 
Safety Endpoints ...................................................................................................................... 26 
3.14.4 
Definition of endpoints ............................................................................................................ 26 

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3.15 
Statistical analysis ............................................................................................................................ 27 
3.15.1 
Demographics and baseline data ............................................................................................ 27 
3.15.2 
Analysis of OS, TTCR, QoL and CE ............................................................................................ 27 
3.15.3 
Analysis of Safety ..................................................................................................................... 29 
4 
Data Management ................................................................................................................................... 29 
4.1 
Quality Control ................................................................................................................................ 29 
4.2 
Data Quality Assurance ................................................................................................................... 29 
4.2.1 
Case Report Forms (eCRFs) ...................................................................................................... 29 
4.2.2 
PET/CT Second opinion board ................................................................................................. 29 
4.2.3 
Monitoring ............................................................................................................................... 29 
4.2.4 
Training of staff ........................................................................................................................ 30 
4.2.5 
Audit and inspections .............................................................................................................. 30 
4.2.6 
Changes in the approved Trial Protocol .................................................................................. 30 
5 
Processing of personal data in the IMPROVE-IT2 study .......................................................................... 30 
6 
Ethics and risk .......................................................................................................................................... 31 
6.1 
Ethics committee approval .............................................................................................................. 31 
6.2 
Blood sampling ................................................................................................................................ 31 
6.3 
Genomic sequence analysis ............................................................................................................. 31 
6.4 
18FDG-PET/CT scan ........................................................................................................................... 32 
7 
Publication and reporting from the trial ................................................................................................. 33 
8 
Economy .................................................................................................................................................. 33 
8.1 
Remuneration to participants ......................................................................................................... 33 
8.2 
Patient compensation scheme (patienterstatningen) .................................................................... 33 
9 
References ............................................................................................................................................... 34 

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1 Introduction 
In Denmark, colorectal cancer (CRC) represents a significant health burden with ~5,000 new cases and ~2000 
CRC related deaths in 20181. 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). 
In Denmark, we have introduced a screening program to improve survival by reducing the number of patients 
diagnosed with synchronous metastases, but poor survival due to disease recurrence remain a major 
problem. If postoperative residual disease could be detected early then survival could be improved. 
Metachronous metastases are most frequently found in the liver and the lungs and if resected with curative 
intent, the 5-year relative survival is ~50-60%.2–5 However, if the metastases are detected too late for 
intended curative treatment and the patients instead receive palliative chemotherapy or best-supportive 
care, then less than 10% are alive after 5 years.2–4,6–8 The ability to resect, as well as the survival after 
resection, are negatively influenced by increasing numbers of metastatic sites and the size of the 
metastases.2,3,5 Thus, early detection of recurrences when tumor burden is low is of paramount value, not 
only to increase the rate of curative resections, but also to increase survival after resection. Local treatment 
of metastases (e.g. radiofrequency ablation) that are small, but non-resectable or potentially resectable in 
patients ineligible for major surgery, have yielded 5-year survival rates up to 40-60%.9–12 This implies that also 
patients considered ineligible to curative resection could benefit from early recurrence detection. 
At time of diagnosis, the majority of patients (75%) present with non-metastatic disease (stage I-III), for which 
standard of care is curative surgery.13 The 5-year relative survival rate for all stage I-III is 80 %, but drops to 
65 % for stage III alone.13 Overall 15-20 % of patients experience recurrence after curative surgery for stage 
I-III CRC with 70-90% detected within 3 years of primary surgery.14–21 Stage III comprises only 37 % of stage I-
III patients13, but account for 70 % of recurrences.22 Due to the high recurrence risk, adjuvant chemotherapy 
is standard of care for stage III patients23 but still ~33% of patients relapse (DK, ~360 stage III 
recurrences/year24).16,18,25 Extending duration of adjuvant chemotherapy beyond the current 6 months offers 
no further reduction in recurrence.26–29 Even 3 months of oxaliplatin-based chemotherapy is sufficient in 
stage III patients with low-risk pathological features.26–29 Consequently, the only alternative option after 
adjuvant chemotherapy is surveillance to enable detection of recurrence, when residual disease has grown 
to a detectable size, but at an early stage that allow curative treatment.30–32 Hence, stage III patients 
represent the optimal focus point for investigation of novel surveillance strategies. 
With current surveillance, only ~15% of recurrences are eligible to curative resection or local treatment33,34 
and the 5-year survival rate for metachronous metastasis is just 9%.33 In Denmark, the national guideline 
recommends CT-scans of thorax and abdomen 12 and 36 months postoperatively, and colonoscopy every 5 
years until age 75.35 Individual centers may offer a slightly more intensive CT-scan surveillance. However, 
similar to previous studies17,36, increasing intensity of CT-based surveillance for stage II-III did not increase 
survival in a large Danish randomized controlled trial.37 More patients do receive intended curative surgery 
for recurrence with intensified surveillance,17 but as more than 80% of patients with stage II-III patients do 
not relapse, this probably dilutes the impact on survival.37 What is needed are methods to identify the ~30 % 
of stage III patients with microscopic residual disease after adjuvant chemotherapy who can benefit from 
high-intensity surveillance.38 
Minimally-invasive blood-based analysis of circulating tumor DNA (ctDNA) is an emerging tool that has this 
potential to identify microscopic residual disease.39–41 It is based on the observation that tumors shed DNA 
fragments into the blood.42 The half-life of ctDNA is less than two hours.43 Consequently, cured patients will 

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not have ctDNA during follow-up, while patients with residual disease are likely to be ctDNA positive. Like for 
all quantification methods, there is a lower limit of detection for ctDNA analysis, and consequently ctDNA 
may not be detectable in patients with very low levels of residual disease e.g. after end of adjuvant 
chemotherapy. However, as the residual disease grows, ctDNA eventually becomes detectable if blood 
samples are analyzed longitudinally. In studies of patients not receiving adjuvant chemotherapy, we and 
others have shown that the risk of relapse in patients with postoperative ctDNA is close to 100%, while the 
risk of relapse in ctDNA negative patients is as low as 10%39–41 (HR=37.66; 95% CI, 4.23 to 335.49; 
P<0.0001).40,41,44 In a recent observational study, we analyzed blood samples collected longitudinally during 
surveillance after adjuvant chemotherapy.45 ctDNA was identified in 13/58 patients of which 92% (12/13) 
relapsed. In multivariate analysis, ctDNA status was a stronger predictor of incipient relapse than other 
predictive factors such as UICC stage, lymphovascular invasion, micro-radical resection status, and 
carcinoembryonic antigen (CEA) (HR, 29.0; 95% CI, 6.4-130; P<0.001)(Figure 1B). Moreover, longitudinal 
ctDNA analysis detected relapse with an average lead time of 8.7 months compared to standard-of-care CT-
imaging (Wilcoxon signed rank test; P<0.001) (Figure 1C). From first ctDNA detection, all plasma samples until 
radiological relapse were ctDNA-positive and revealed a 50-fold increase in the mean ctDNA level, indicating 
that the tumor burden increased dramatically while the patients awaited radiological detection of the relapse 
(Figure 1D). Hence, ctDNA surveillance may detect relapse sufficiently early to change patient management. 
Combining ctDNA and radiological assessments could result in earlier detection of recurrent disease and 
identify more patients eligible for curative treatment and generally enable early implementation of local and 
systemic therapies. This will likely improve treatment and survival, as one of the fundamental principles of 
oncology is that it is more effective to treat few and small lesions than multiple and large lesions.46 
 
 
Figure 1 – Longitudinal ctDNA analysis after adjuvant chemotherapy enables early detection of relapse. (A) Proposed IMPROVE-IT2 
trial comparing standard-of-care radiological surveillance to ctDNA guided surveillance. (B) Relapse risk after adjuvant chemotherapy 
stratified by post-adjuvant ctDNA status. Patients were scored ctDNA positive if they at any time-point post-adjuvant were positive 

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and negative if they at all post-adjuvant time-points were negative. (C) Comparison of time to relapse by ctDNA analysis and standard-
of-care CT imaging. The average lead time of ctDNA was 8.7 months. (D) For all relapsing patients, the ctDNA levels in plasma 
increased over time from ctDNA detection to radiological detection.  
2 Objectives 
The overall objective of this study is to establish a randomized controlled trial investigating the benefit of 
ctDNA-guided postoperative surveillance for stage III and stage II high-risk colorectal cancer (CRC) patients 
compared to the current standard-of-care CT-scan surveillance (Figure 1A). Currently, only ~15% of 
recurrences are detected sufficiently early to enable curatively intended intervention. The central hypothesis 
of this study is that ctDNA-guided postoperative surveillance will lead to shorter time to recurrence 
detection, which will allow curatively intended intervention in time to improve overall survival.  
2.1 Primary Objective 
The primary objective is to investigate if ctDNA-guided post-operative surveillance to guide radiological 
assessments will result in a higher fraction of recurrence patients receiving curative intended or local 
metastasis-directed treatment for CRC recurrence as compared to current Danish surveillance strategy 
within 3 years after primary surgery.   
2.2 Key Secondary Objectives - Oncological 
2.2.1 Key Secondary Objective 1 (S1)  
To investigate if ctDNA-guided surveillance is associated with a shorter time from randomization to clinical 
recurrence (TTCR) than standard-of-care surveillance. 
2.2.2 Key Secondary Objective (S2)  
To investigate if 3- and 5-year overall survival (OS) after surgery is non-inferior with ctDNA guided 
surveillance compared to current standard-of-care CT-based surveillance. To take immortal time, typically 
4-8 months, from the time of surgery until randomization into account, overall survival will be estimated 
with two different starting points of follow-up: 1) from time of surgery and 2) from time of randomization. 
For both starting points, OS will be assessed at 3- and 5-years after surgery.  
2.3 Key Secondary Objectives – Quality of Life 
2.3.1 Key Secondary Objective 3 (S3) 
To compare the quality of life (EORTC QLQ-C30 and EQ-5D-L5), fear of cancer recurrence inventory (FCRI), 
and impact of events scale for cancer (IES-C) for patients following ctDNA guided and the standard-of-care 
surveillance. 
2.4 Secondary Objectives 
2.4.1 Secondary Objective 4 (S4) 
To assess the cost-effectiveness of ctDNA-guided and standard-of-care surveillance, to provide decision 
support for clinicians and other decision-makers. 
2.4.2 Secondary Objective 5 (S5)  
To assess, and compare, the protocol adherence rates (AR) for patients following ctDNA-guided and 
standard-of-care surveillance.  

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2.4.3 Secondary Objective 6 (S6)  
To assess if the time to molecular recurrence (TTMR), i.e., detection of ctDNA- and/or elevated level of 
Carcinoembryonic antigen (CEA) are similar, for patients following ctDNA guided and patients following 
standard-of-care surveillance.  
2.4.4 Secondary Objective 7 (S7)  
To describe any changes in quality of life (EORTC QLQ-C30 and EQ-5D-L5), fear of cancer recurrence inventory 
(FCRI), and impact of events scale for cancer (IES-C) from time of positive ctDNA until time of clinically verified 
recurrence.  
2.4.5 Secondary Objective 8 (S8)  
To investigate if ctDNA growth rate analyses, performed on blood samples collected within a short interval 
(2-3 weeks apart), can stratify ctDNA-positive patients into groups of fast and slow-growing tumors. 
2.4.6 Secondary Objective 9 (S9)  
To compare the rate of clinical recurrence detection during the time intervals: randomization-12 months 
after surgery, 13-24 months after surgery, and 25-36 months after surgery for patients with ctDNA guided 
and standard-of-care surveillance. 
2.4.7 Secondary Objective 10 (S10)  
To compare the cumulative incidence of clinical recurrence at 12 months, 24 months, and 36 months after 
surgery for patients with ctDNA-guided and standard-of-care surveillance. 
3 Investigational plan 
3.1 Overall study design 
The study will establish the randomized clinical trial IMPROVE-IT2 investigating the benefit of ctDNA guided 
surveillance for stage III and stage II high-risk CRC patients treated with adjuvant chemotherapy, compared 
to the current one-size fits all surveillance. 
CRC patients treated with adjuvant chemotherapy will be randomized into two arms: 
• 
The experimental arm: ctDNA analysis will be performed at months 4, 8, 12, 16, 20 and 24 post-
operatively. ctDNA analysis terminates at month 24, because all relapse patients in our previous 
studies tested ctDNA positive no later than month 18 (Figure 1C). At time of first positive ctDNA, 
patients undergo a comprehensive radiological assessment with a whole-body PET/CT-scan. A 
colonoscopy will be performed if the PET/CT-scan is without evidence of recurrence. If the initial 
assessment is without evidence of recurrence, patients will be offered intensive radiological 
surveillance (PET/CT-scans every 3 months, until recurrence detection or 21 months has passed). 
Radiological follow-up is ended after 21 months, because the maximal lead time of ctDNA to CT-scan 
was 16.5 months in our previous prospective studies (Figure 1C). ctDNA negative patients will in 
addition to blood-sampling only receive an end-of-study CT-scan. This will be performed between 
months 36 and 37. A blood sample will be collected at the same time-point for molecular reference. 
 
• 
The control arm: Patients will receive surveillance according to current Danish guidelines with CT-
scans at months 12 and 36. Longitudinal blood samples will be collected at the same time-points as 
in the ctDNA surveillance arm but will not be analyzed until the end of the trial. They serve to enable 
post-trial comparison of oncological outcomes for the two arms stratified for ctDNA-status. 

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Quality of life (QoL) will be monitored in all patients, independent of randomization, by use of the validated 
Danish version of the European Organization for Research and Treatment of Cancer Quality of Life 
questionnaire, Core 30 (EORTC QLQ-C30 ver. 3.0), Fear of Cancer Recurrence Inventory (FCRI), Impact of 
Events Scale for Cancer (IES-C) and European Quality of Life – 5 Dimensions (EQ-5D-5L).  
Further, ctDNA positive patients (in the experimental arm) will be asked to complete the QLQ-C30, FCRI, IES-
C and EQ-5D-5L questionnaires before each PET-CT scan. Patients will be their own controls. It is an 
explorative, longitudinal study. 
3.2 Study timetable 
Start inclusion: January 2020 
End inclusion:  December 2023 
End data collection: December 2028 
Primary endpoint reached, ready to publish: December 2025 
Follow-up: 5 years 
Last patient last visit:  December 2026 
3.3 Setting 
The randomized trial “IMPROVE-IT2” is an independent sub-study of the ongoing prospective multicenter 
ctDNA study IMPROVE (ClinicalTrials.gov: NCT03637686, Committees on Biomedical Research Ethics in the 
Central Region of Denmark j. no. 1-10-72-3-18). IMPROVE-IT2 is registered with ClinicalTrial.gov: 
NCT04084249. During 2018-2020, IMPROVE will recruit 1,800 stage I-III CRC patients at the following major 
surgical departments in Denmark (Aalborg, Herning, Viborg, Randers, Aarhus, Horsens, Odense, Svendborg, 
Herlev, Bispebjerg, Roskilde, Slagelse og Køge). Part II of the IMPROVE study is an observational trial involving 
longitudinal ctDNA analysis of stage III and stage II high-risk patients. However, because of the convincing 
evidence supporting the clinical potential of ctDNA for early relapse detection, recently provided by us 40,45,47 
and others41,48–51, we have established IMPROVE-IT2 with the intention to progress from the observational 
to interventional setting. While IMPROVE-IT2 is recruiting, we will invite stage III and stage II high-risks 
patients treated with adjuvant chemotherapy to participate in IMPROVE-IT2, rather than IMPROVE part II.  
3.4 Sample size 
Assessment of the sample size needed to meet the Primary Objective (see 2.1) 
Based on the first 6 months of enrollment in IMPROVE, it is expected that 385 stage III patients receiving 
adjuvant therapy will be approached in the recruitment period for IMPROVE IT2. Our current recruitment 
rate to the IMPROVE longitudinal blood sample study is 66%. Meaning that 254 patients are likely to 
participate (127 in each arm). The recurrence rate of this patient population is 33% (n=84). Hence, with a 1:1 
randomization we expect 42 recurrences in each arm. Primary endpoint of the study is the fraction of patients 
with recurrent, metastatic disease receiving curative-intent resection or local treatment. With standard-of-
care surveillance, approximately 15% of relapse events are eligible to curative-intent treatment. Assuming 
this fraction can be increased by a factor of 3 (to 45%) in the experimental arm. In order to achieve a power 
of 80% to detect the difference, at a 5% significance level, a sample size providing 35 recurrence patients in 
each arm is needed, which is below the number available, even when assuming a 10% drop-out rate.  
UPDATED SAMPLE SIZE (MAY 2022) 

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Our new unpublished study on updated recurrence rates has disclosed, that the recurrence rates in more 
recent years have decreased to approximately 25 % (compared to 33 % in the above calculation). This means 
that our power calculation no longer applies. Instead, we will be aiming at recruiting 310 patients (155 in 
each arm). With an estimated recurrence rate of 25 % (n = 78), we expect to have 39 recurrences in each 
arm. Even with a drop-out rate of 10 % this will be enough to reach the sufficient power (35 recurrences in 
each arm, see above). We have recruited 82 patients in 2020, 88 patients in 2021 and up to now (May) we 
have recruited 50 patients in 2022, making a total of 220 patients. Recruiting another 90 patients will 
estimated take 1 year, meaning that it is feasible to reach the new aimed sample size by prolonging the 
recruitment period with 1 year. Furthermore, we are observing a drop-out rate of approximately 10 % which 
fits the estimated rate in the power calculation.    
UPDATED SAMPLE SIZE (NOVEMBER 2022) 
We are experiencing a larger drop-out rate than expected. Currently, we have a drop-out rate of 14 %, 
compared to the 10 % that we were expecting. To compensate for this, we need to update our power 
calculation. We want to increase the aimed number of recruited patients to 340. With an expected 
recurrence rate of 25 % (n = 85), this will lead to approximately 42 recurrences in each arm. Even with a 
dropout rate of 15 %, we will be able to reach sufficient power (at least 35 recurrences in each arm, see 
above). We are currently recruiting approximately 10 patients each month and have a total of 272 patients 
in the study, meaning that we will be able to reach the new aimed recruitment within the planned study 
period. 
Assessment of the sample size needed to meet the secondary objective S3 related to Quality of Life (See 
2.2.2) 
A mean global health-status/QoL difference of 10 points is defined to be the threshold of clinical 
relevance.52,53 Assuming a mean global health-status/QoL score of 73 with a standard deviation of 2353, then 
with no difference between experimental and control arm, 91 patients in each arm are required to show with 
a power of 90% that the lower limit of a one-sided 95% confidence interval will be above the non-inferiority 
limit of -10. 
3.5 Inclusion of patients  
The patients eligible for the IMPROVE-IT2 study will be identified at the treating surgical department, and 
must meet the criteria described in the below section.  
3.5.1 IMPROVE-IT2 patient criteria 
3.5.1.1 
Inclusion criteria 
• 
Participation in IMPROVE (Committees on Biomedical Research Ethics in the Central Region of 
Denmark j. no. 1-10-72-3-18) 
• 
Colon or rectal cancer, tumor stage III (pT1-4N1-2,cM0) or stage II high-risk (pT4N0,cM0 and pT3N0, 
cM0 with either of the risk factors (<12 lymph nodes, anastomosis leakage, emergency surgery, signet 
ring adenocarcinoma) described in the national guideline for adjuvant therapy to stage II cancer) 
• 
Has received curative-intent resection and is a candidate for adjuvant chemotherapy (3 or 6 months 
regime) 
3.5.1.2 
Exclusion criteria 
• 
Not treated with adjuvant chemotherapy (patients who receive incomplete treatment are NOT 
excluded, as the analysis is based on intention to treat) 

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• 
Synchronous colorectal and non-colorectal cancer diagnosed per-operatively (except skin cancer 
other than melanoma) 
• 
Other cancers (excluding colorectal cancer or skin cancer other than melanoma) within 3 years from 
screening 
• 
Patients who are unlikely to comply with the protocol, inability to return for subsequent visits, unable 
to have blood samples drawn and/or otherwise considered by the Investigator to be unlikely to 
complete the study 
If a patient is excluded during the trial, it is the responsibility of the treating department to make sure that 
the patient is reenrolled in a surveillance program in accordance with national standards and guidelines. 
3.5.2 Time of inclusion and allocation procedure 
Two-stage procedure: 
1. Day 14 post-OP visit (Baseline): (when patients are informed about the pathology of the resected tumor). 
Patients with stage III or high-risk stage II CRC, who are referred to an oncological department for adjuvant 
chemotherapy, are informed about the IMPROVE-IT2 trial and informed consent will be obtained. The signed 
consent form can be collected both at the Day 14 and Month 4 visits.  
 
2. Post-OP month 4: (Time of first blood-sample in IMPROVE-IT2): 
Patients, who have not received any adjuvant chemotherapy, are excluded from IMPROVE-IT2. 
Patients, who have received adjuvant chemotherapy, will be allocated to either ctDNA guided surveillance 
(experimental arm) or standard-of-care surveillance (control arm) using a concealed web-based 
randomization service managed by Aarhus University (REDCap) (See section 3.6). 
 
Recruitment of patients already participating in IMPROVE part II: 
Patients, who at the time of initiation of the IMPROVE-IT2 trial already are included in the ongoing 
observational IMPROVE part II surveillance study (See the protocol associated with j. no. 1-10-72-3-18 the 
Committees on Biomedical Research Ethics in the Central Region of Denmark), can be included in IMPROVE-
IT2 before completion of adjuvant chemotherapy.  
3.5.3 Procedures for patient recruitment and informed consent 
Patients, who meet the inclusion criteria are approached in person at the treating surgical department before 
completion of adjuvant therapy. Here the patients are given written information about the project and it is 
arranged when verbal information about the project will be given. The patients are encouraged to bring an 
assessor to this session. The oral information is given by trained health care professionals (either the surgeon 
or specially trained project nurses). Oral information is given in a closed room to ensure that the conversation 
can take place undisturbed. Besides information about the project, the patients are also informed about their 
rights as participants in a research project. Furthermore, as the project involves genomic sequencing, which 
may disclose genetic variants predisposing to specific diseases, the participants are also offered genetic 
counseling before making their decision to participate. After the oral information has been given, the patients 
are allowed at least 24 hours to consider their decision to participate before they are approached in order to 
obtain written informed consent. Informed consent has to be obtained prior to commencement of any study-
related procedure. 
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. 

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The written participant information contains contact information to the person being primary responsible 
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, without this having any impact on the relationship 
to the department, or on current or future treatment. It also mentions that study involves a biobank, and 
that residual biological 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 approved 
by the Danish Data Protection Agency (j. no. 1-16-02-27-10). 
3.5.4 Patient information and consent 
The patient information text and consent form is available as Appendix A.  
3.6 Randomization 
Randomization will be performed at each clinical site using a concealed centralized web-based randomization 
service managed by Aarhus University (REDCap). 
 
Randomization 1:1 into two groups 
1. ctDNA guided surveillance without routine CT-scans (experimental arm) 
2. Standard-of-care surveillance with routine CT-scans (control arm) 
 
Using a stratified block randomization algorithm, with block size 4. Stratification is based on three variables: 
1. Location of primary tumor:  right-sided colon incl. right flexure, transverse/left-sided 
colon, rectum 
2. Tumor stage: pT3-4, pN0 and pT1-3, pN1 versus pT4, pN1 and pT1-4, pN2  
3. Standard-of-care surveillance program intensity: sites following the national 
recommendation with 12 and 36 months CT-scans versus sites with more intensive 
surveillance)  
 
We stratify for variables 1 and 2 with the aim to balance the recurrence rate in the two groups. The variables 
were chosen as it was assessed that they were most significantly associated with risk, location and prognosis 
of recurrence 
We stratify for variable 3 with the aim to balance patients from sites with high (or low) intensity standard-of-
care surveillance between the groups. This is important as most recruitment sites have low intensity 
standard-of-care surveillance and we want to avoid unbalanced randomization of patients from high-
intensity sites. 
 
3.7 Quality of Life baseline consideration: 
The QoL of the individual patients may vary considerably after completion of adjuvant therapy. A very poor 
QoL of single individuals could endure for duration of the follow-up irrespectively of which group they are 
allocated to. To avoid that potential unbalances in patient QoL outlier distributions affect the QoL 
comparison between the surveillance groups, it is important to have a common baseline starting point for 
the QoL assessments. This will enable evaluation of differences in QoL between groups, by making the QoL 
measurements in each patient relative to the patient’s own baseline value. 

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Baseline QoL is measured after end of adjuvant chemotherapy and before the patient and project 
personnel is informed about the allocated surveillance program. Thereby, we ensure the baseline 
assessment is unbiased.    
 
3.8 Study flow 
The time-schedule and procedures for inclusion and data collection at baseline (Day14) and during follow-up 
are described below and outlined in flow charts below. The specific procedures regarding blood sampling, 
handling and analysis will be described separately. 
Flowchart: Control arm and experimental arm (ctDNA negative) 
  
Day14 
(Baseline) 
Mo3 
Mo4 
Mo7 
Mo8 Mo12 Mo16 
Mo18 
Mo20 
Mo24 
Mo36 
Mo36-
Mo38 
Patient 
inclusion 
  
  
  
  
  
  
  
  
  
  
  
  
Patient 
exclusion 
  
If ACT 
was not 
initiated 
  
  
  
  
  
  
  
  
  
  
Informed 
consent 
  
  
  
  
  
  
  
  
  
  
  
  
Registration of 
baseline data 
  
  
  
  
  
  
  
  
  
  
  
  
Adjuvant 
chemotherapy 
(ACT) data 
registration 
Record 
intention 
to 
administer 
ACT 
Record 
if ACT 
was 
initiated 
or not, 
and the 
planned 
duration 
If 3 
mo 
ACT, 
record 
end 
date  
If 6 
mo 
ACT, 
record 
end 
date 
  
  
  
  
  
  
  
  
Randomization 
  
  
  
  
  
  
  
  
  
  
  
  
CT scan 
  
  
  
  
  
Control 
arm 
only 
  
  
  
  
  
  
QoL 
questionnaires 
(QLQ-C30, 
FCRI, IES-C, 
EQ-5D-5L) 
  
  
If 6 
mo 
ACT; 
send 
out at 
mo7 
  
  
  
  
  
  
  
  
  
Blood 
sampling 
  
  
If 3 
mo 
ACT; 
draw 
blood  
after 
ACT 
end 
  
If 6 
mo 
ACT, 
draw 
blood 
after 
ACT 
end 
  
  
  
  
  
  
  
Flowchart: Experimental arm (ctDNA positive)  
  
Every 3 months 
until radiological 
recurrence or up to 
21 months 
Mo12 
Mo36 
Mo36-Mo38 

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CT scan 
  
  
If no recurrence was 
detected during 21 
months of PET-CT 
  
PET-CT scan 
  
  
  
  
Colonoscopy 
Performed if the first 
PET/CT-scan is 
without evidence of 
recurrence 
  
  
  
QoL questionnaires (QLQ-
C30, FCRI, IES-C, EQ-5D-
5L)QoL questionnaires 
(FCRI, IES-C, and EQ-5D-
5L) 
  
  
  
  
Blood sampling 
  
  
If no recurrence was 
detected during 21 
months of PET-CT 
  
 
 
Post OP day 14 visit (inclusion visit, Baseline)  
• 
Patients full filling the inclusion criterias are informed about IMPROVE-IT2 and asked to consent to 
participation 
• 
Patients informed about the pathology report 
• 
Decision about referral to oncology department for adjuvant chemotherapy according to standard 
guidelines and patient’s preferences 
• 
Inform patients that IMPROVE-IT2 randomization will be conducted after adjuvant treatment has 
commenced. 
• 
Inform patients if they do not receive adjuvant treatment, they will be excluded from IMPROVE-IT2, 
and instead offered participation in the observational study IMROVE part II surveillance. 
• 
Inform patients that they as part of the trial will be asked to fill in questionnaires about their quality 
of life before start in the study (after end of adjuvant treatment), and again at 12, 18, and 24 months 
and after end of surveillance. Inform patients that access to the QoL questionaires will be sent to 
them by digital mail (e-boks). 
• 
Record basic information on all patients invited to participate (CRF Invitation) 
o CPR number on all invited patients 
o whether they accepted participation or not 
o whether the patients accepted or declined to receive information about incidental genomic 
findings with potential health consequences  
• 
Record baseline patient information (CRF IMPROVE-IT2 BASELINE) 
o WHO performance status 
o Height, weight and Body Mass Index (BMI) 
o Smoking and alcohol use 
o Imaging used for pre-operative work-out 
o If complete colonoscopy or Computed Tomography (CT) colon has been made or is scheduled 
post-operatively 
o diagnosis of other cancers within 3 years from screening 
• 
Record intention to administer adjuvant chemotherapy (CRF IMPROVE-IT2 ADJ1) 
• 
Record surgery and pathology data in the CRF (CRF IMPROVE-IT2 PATHOLOGY)  

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3.8.1 Post OP month 3 (Status of adjuvant therapy, EXCLUSION of patients NOT treated with 
adjuvant therapy, RANDOMIZATION of patients treated with adjuvant therapy, send QoL to 
patients expected to end adjuvant therapy before month 4)  
• 
Record in information about adjuvant chemotherapy (CRF IMPROVE-IT2 ADJ2) 
o If adjuvant therapy was initiated (yes or no) 
o Adjuvant therapy start date 
o Planned regimen 
o Planned duration: 3 months or 6 months 
• 
For patients who received adjuvant chemotherapy: 
o Check the patient file if adjuvant therapy has ended or not. Record the end date or the 
expected end date (CRF IMPROVE-IT2 ADJ3). 
o Call the patient to 
▪ 
Confirm status of adjuvant chemotherapy, the end date or expected end date.  
▪ 
Decide with the patient the date for the 4 month visit and blood sample 
collection.  
• 
For patients with adjuvant therapy “end date” or “expected end date” 
before month 4 (plus 14 days) make sure the visit is scheduled after the 
end of chemotherapy (at the earliest 10 days after).  
▪ 
For patients were adjuvant therapy has been, or is expected to end, before the 
4 month visit:  
• 
Inform that a link to an online QoL questionnaire will be sent by digital 
mail (e-boks) and that this has to be completed after end of adjuvant 
chemotherapy and before the 4 month visit. The project personnel, shall 
ask if the patient has an digital e-boks. If not, then a paper version of the 
questionnaire shall be sent by “Quick” mail. 
• 
Complete the CRF IMPROVE-IT2 QOL-TRIGGER, which will trigger 
REDCap to send a questionnaire consisting of EORTC QLQ-C30, FCRI, IES-
C and EQ-5D-5L to the patients. 
▪ 
For patients whose adjuvant therapy regimen is NOT expected to end before the 
4 month visit: 
• 
The 4 month visit is only to collect blood 
• 
NOTE: These patients will be approached in month 7 to record the end 
date of adjuvant therapy and distribute the QoL questionnaires.  
• 
For patients NOT treated with adjuvant therapy: 
o Call the patient to: 1) inform that the patient is EXCLUDED from IMPROVE-IT2 (complete 
CRF IMPROVE-IT2 END OF STUDY) and 2) inform about the very similar, but 
observational, study IMPROVE part II and ask the patient to consent to participate in this.  
• 
Patients TREATED adjuvant chemotherapy are RANDOMIZED to either ctDNA guided surveillance 
(experimental arm) or standard surveillance (control arm). Randomization is performed in 
REDCap after the 3 month phone call to the patient. Randomization result is not revealed to 
patient nor project personnel at this time point.  
o Patients whose adjuvant therapy has ended before month 4 are informed at the 4 month 
visit.  
o Patients whose adjuvant therapy ends after month 4 are informed at the 8 month visit. 

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For the post OP month 4 visit, the handling of patients randomized to both the experimental and 
control arm are described together. Thereafter, the two arms are described separately. 
 
3.8.2 Post OP month 4 visit (collection of a blood sample, and information about randomization) 
• 
Register status of adjuvant therapy (CRF IMPROVE-IT2 ADJ3) 
o Register if adjuvant therapy has ended or not 
o If adjuvant therapy has ended, record end date 
o If adjuvant therapy has not ended, then check if expected end date should be updated 
• 
Patients who are still receiving adjuvant therapy are informed that they receive the QoL-
questionnaire and randomization result after end of adjuvant therapy.  
• 
For patients who were sent the QoL questionnaire at post-OP month 3 (section 3.7.1) the project 
personnel will check if the QoL questionnaire has been completed. If not the project personnel 
will assist the patients in doing so on site.   
• 
Randomization result: Will first be reveal to the personnel and patient at completion of the QoL- 
questionnaire. Only patients whose adjuvant therapy has ended are informed about their 
randomization allocation and the associated follow-up plan. 
• 
Blood sampling is performed on all patients. 
• 
For patients in the experimental arm where adjuvant therapy has ended, the blood is analyzed 
for ctDNA immediately. The result is registered in the IMPROVE-IT2 REDCap database by 
Department of Molecular Medicine 
o ctDNA negative result: patients will be informed about the result by secure digital mail 
via E-boks, and informed that they will be contacted again in due time before Post OP 
month 8 visit. 
o ctDNA positive result: patients will be informed about the result at a visit in the 
outpatient clinic and at the same time informed about the subsequent follow-up (See 
section 3.8.5.2.2). 
• 
For all other patients the blood will be stored and analyzed at end of study (patients in the control 
arm and patients in the experimental arm still receiving adjuvant therapy). 
3.8.3 Post OP month 7 (Status of adjuvant therapy for all patients who were still in active treatment 
at the month 4 visit)  
• 
Check the patient file to record the end date or the expected end date (CRF IMPROVE-IT2 ADJ3). 
• 
Call the patient to 
o Confirm status of adjuvant chemotherapy, the end date or expected end date.  
o Decide with the patient the date for the 8 month visit.  
▪ 
Make sure the visit is scheduled after the end of chemotherapy (at the earliest 
10 days after). 
o Inform that a link to an online QoL questionnaire will be sent by digital mail (e-boks) and 
that this has to be completed after end of adjuvant chemotherapy and before the 8 
month visit. The project personnel, shall ask if the patient has an digital e-boks. If not, 
then a paper version of the questionnaire shall be sent by “Quick” mail. 
o Complete the CRF IMPROVE-IT2 QOL-TRIGGER, which will trigger REDCap to send a 
questionnaire consisting of EORTC QLQ-C30, FCRI, IES-C and EQ-5D-5L to the patients. 

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3.8.4 Post OP month 8 visit 
• 
For patients who were still in active treatment at the month 4 visit 
o Record end date of adjuvant chemotherapy for those with only expected end day at Post 
OP month 7 call. (update CRF IMPROVE-IT2 ADJ3) 
o The project personnel will check if the QoL questionnaire has been completed. If not the 
project personnel will assist the patients in doing so on site.   
o Randomization result: After the completion of the QoL questionnaire the patients and 
personnel are informed about the randomization allocation. The patients are informed 
about the associated follow-up plan. 
 
• 
All patients: Now adjuvant therapy has ended for all patients, and they have all been informed 
about their randomization group and will receive follow-up accordingly 
o Blood sampling is performed on all patients. 
o For patients in the experimental arm the blood is analyzed for ctDNA immediately. The 
result is registered in the IMPROVE-IT2 REDCap database by Department of Molecular 
Medicine 
▪ 
ctDNA negative result: patients will be informed about the result by secure 
digital mail via E-boks, and informed that they will be contacted again in due 
time before Post OP month 12 visit. 
▪ 
ctDNA positive result: patients will be informed about the result at a visit in the 
outpatient clinic and at the same time informed about the subsequent follow-
up (See section 3.8.5.2.2). 
• 
For patients in the control arm the blood will be stored and analyzed at end of study. 
 
3.8.5 Longitudinal post OP visits (mo12 to mo36) 
3.8.5.1 
For patients in the Control arm: 
• 
Longitudinal PostOP blood sampling (mo12, mo16, mo20, mo24 and mo36) 
• 
Follow-up: At months 12 and 36 project personnel records the results from the standard CT-scans 
performed at 12 and 36 months (CRF CTSCAN 12mo, CRF CTSCAN 36mo) 
• 
Imaging because of suspicion of recurrence: At month 36 the hospital files are assessed for 
additional imaging events, and related pathology and treatment notes, performed due to 
suspicion of recurrence (CRF-CTSCAN-Suspicion). 
• 
Quality of life at months 12, 18, 24 and 38: Before the blood collection visits at months 12 and 
24, and at month 18 (between the blood collection visits at months 16 and 20), and again after 
end of the surveillance program (after the month 36 CT-scan and before month 38), a request to 
complete the online QoL questionnaire (EORTC QLQ-C30, FCRI, IES-C, and EQ-5D-5L) will be sent 
electronically to the patients via REDCap (CRF IMPROVE-IT2 QOL-TRIGGER). Patients without an 
e-boks will get the QoL questionnaires in paper form by regular mail and project personnel will 
register the results in REDCap manually.  
• 
Mo12 and mo24 QoL questionnaire completion check: in relation to the subsequent blood 
collection visits (mo 12 and mo 24) the project personnel will check if the QoL questionnaire has 
been completed. If not, the project personnel will assist the patients in doing so on site. If this is 
not possible, the project personnel hand over paper versions and register the results in REDCap 
manually. 

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• 
Mo18 and mo38 QoL questionnaire completion check: As these time points are not related to 
hospital visits, project personnel will check if the QoL questionnaire has been completed 2 weeks 
after being submitted. If not the project personnel will trigger a digital reminder, which will be 
send to the patient (CRF IMPROVE-IT2 QOL-TRIGGER). Patients without e-boks, who has not 
completed the questionnaire, will be contacted by phone. 
 
3.8.5.2 
For patients in the Experimental arm 
3.8.5.2.1 Experimental arm, ctDNA negative: 
• 
Longitudinal PostOP blood sampling for ctDNA guided surveillance (mo12, mo16, mo20 & mo24) 
o blood is analyzed for ctDNA immediately. The result is registered in the IMPROVE-IT2 REDCap 
database by Department of Molecular Medicine. 
o ctDNA negative result: the patients are informed about the result by secure digital mail via 
E-boks. In due time, the patients are informed about the next post OP blood sampling visit  
o ctDNA positive result:  See section 3.8.5.2.2 
• 
End of study CT-scan and blood collection: after month 36 and before month 37, the patients are 
invited for a standard CT-scan. The result is recorded in the REDCap database (CRF-
CTSCAN36mo). A blood sample will also be collected at this visit. The blood sample will be stored 
in the biobank and analysed at end of study. 
• 
Quality of life at months 12, 18, 24 and 38: Before the blood collection visits at months 12 and 
24, and at month 18 (between the blood collection visits at months 16 and 20), and again after 
end of the surveillance program (after the month 36 CT-scan and before month 38), a request to 
complete the online QoL questionnaire (EORTC QLQ-C30, FCRI, IES-C, and EQ-5D-5L) will be sent 
electronically to the patients via REDCap (CRF IMPROVE-IT2 QOL-TRIGGER). Patients without an 
e-boks will get the QoL questionnaire in paper form by regular mail and project personnel will 
register the results in REDCap manually.  
• 
Mo12 and mo24 QoL questionnaire completion check: in relation to the subsequent blood 
collection visits (mo 12 and mo 24) the project personnel will check if the QoL questionnaire has 
been completed. If not, the project personnel will assist the patients in doing so on site. If this is 
not possible, the project personnel hand over paper versions and register the results in REDCap 
manually. 
• 
Mo18 and mo38 QoL questionnaire completion check: As these time points are not related to 
hospital visits, project personnel will check if the QoL questionnaire has been completed 2 weeks 
after being submitted. If not the project personnel will trigger a digital reminder, which will be 
send to the patient (CRF IMPROVE-IT2 QOL-TRIGGER). Patients without e-boks, who has not 
completed the questionnaire, will be contacted by phone. 
• 
Imaging because of suspicion of recurrence: At month 36 the hospital files are assessed for 
additional imaging events, and related pathology and treatment notes, performed due to 
suspicion of recurrence (CRF-CTSCAN-Suspicion). 
3.8.5.2.2 Experimental arm, ctDNA positive: 
• 
PET-CT follow-up plan: Patients will be informed about the positive ctDNA result, at a visit in the 
outpatient clinic and at the same time informed about the change in follow-up. The new follow-
up entails PET-CT scans every third months until the residual disease has been detected, or 21 
months has passed.  
o The first PET-CT scan shall be performed immediately after the outpatient visit.  

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o If the PET-CT scan is negative, the scan will be reviewed by a second opinion board to evaluate 
the technical quality and the finding (4.2.2). 
o All PET-CT scans (positive or negative findings) will be discussed on the local site MDT-
conference (CRF IMPROVE-IT2 MDT).  
▪ 
If the local site MDT conference agrees that residual disease has been identified, the 
subsequent treatment will be at the discretion of the MDT.  
• 
The site must upload to the IMPROVE-IT2 REDCap database, a scan of the 
MDT records and, if relevant, other documents describing the final 
treatment decision. 
▪ 
If the MDT concludes that the finding is negative or inconclusive, then the patient 
will be invited for a new PET-CT scan every 3 months according to the PET-CT follow-
up plan. 
▪ 
After the first negative PET-CT scan, the patient will be offered a colonoscopy. 
  
o The results of PET-CT-scans are recorded in the IMPROVE IT2 REDCap database (CRF PET 
0mo, CRF PET 3-21mo) 
• 
Longitudinal blood sampling: blood is collected in relation to each PET-CT scan. These samples 
will not be analyzed before end of study. 
• 
Quality of life: Before each PET-CT scan a questionnaire (EORTC QLQ-C3, FCRI, IES-C and EQ-5D-
5L) will be sent to the patients electronically from REDCap (CRF IMPROVE-IT2 QOL-TRIGGER). 
Patients without an e-boks will get the QoL questionnaires in paper form by regular mail and 
project personnel will register the results in REDCap manually. 
• 
At the hospital visits project personnel will check if the QoL questionnaires have been completed. 
If not, the project personnel will assist the patients in doing so on site. If this is not possible, the 
project personnel hand over paper versions and register the results in REDCap manually.  
• 
The collection of QoL assessments stops, when recurrence has been diagnosed. 
• 
At end of study (between months 36 and 38) all ctDNA positive patients in the experimental arm 
are requested to complete the online QoL questionnaire (EORTC QLQ-C30, FCRI, IES-C and EQ-
5D-5L). This will be sent electronically to the patients from REDCap (CRF IMPROVE-IT2 QOL-
TRIGGER). Patients without an e-boks will get the QoL questionnaire in paper form by regular 
mail and project personnel will register the results in REDCap manually. As this time point is not 
related to a hospital visit, project personnel will check if the QoL questionnaire has been 
completed 2 weeks after being submitted. If not the project personnel will trigger a digital 
reminder, which will be send to the patient (CRF IMPROVE-IT2 QOL-TRIGGER). Patients without 
e-boks, who has not completed the questionnaire, will be contacted by phone. 
• 
End of study CT-scan: At month 36, patients who has completed 21 months of PET-CT follow-up 
without detection of recurrence are invited for a standard CT-scan. The result is recorded in the 
REDCap database (CRF CTSCAN 36mo). 
• 
Imaging because of suspicion of recurrence: At month 36 the hospital files are assessed for 
additional imaging events, and related pathology and treatment notes, performed due to 
suspicion of recurrence (CRF-CTSCAN-Suspicion). 
3.9 CT-scan standard 
All CT-scans will be made in accordance with the national standard. This is the responsibility of the treating 
department.  

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3.10 PET-CT-scan technical standard 
The quality of the PET-CT scans performed in the study should comply to the pre-specified minimum 
technical standard described in Appendix B. See also section 4.2.2, about the PET-CT second opinion board. 
3.11 Collection, handling, and biobanking of biological samples 
3.11.1 Collection and handling of blood samples 
Collection of blood samples is performed in collaboration with Regionernes Bio- og Genom Bank (RBGB). At 
each blood draw, a total of 80 mL blood is collected. Plasma, serum, and buffy coat (nucleated blood cells) 
are extracted from the blood. I addition to the 80 mL blood donation via syringe, ctDNA positive patients 
(see section 3.8.5.2.2) will be asked to donate a maximum of 24 droplets of blood (50-400 uL) to a blood 
spot, by pricking the finger with a lance. The blood spot sampling is optional. 
Drawing, handling and storage of the blood is done in accordance to a standard operating procedure 
(SOP)(Appendix C). From the extracted plasma circulating-free-DNA will be extracted and analyzed for 
presence of tumor associated somatic mutations. The serum will be used for measurement of 
carcinoembryonic antigen (CEA), which is the only approved blood based biomarker for monitoring tumor 
burden. The CEA marker will be used as a reference for the performance of ctDNA as a marker of tumor 
burden. From the nucleated blood cells constitutional DNA will be extracted. This DNA will be used as a 
reference when identifying the tumor associated somatic mutations in the circulating free DNA (cfDNA) and 
in the tumor DNA. cfDNA will be extracted from blood spots and will be analyzed for the presence of ctDNA. 
The tumor and normal mucosa tissue biopsies will be collected as part of the IMPROVE trial. 
3.11.2 Research Biobank for the IMPROVE study:  
All blood samples will be stored in a research biobank associated with the IMPROVE study (Danish Data 
Protection Agency approval j. no.: 1-16-02-968-17). Patients will only be included if they consent in writing 
to collection and biobanking of their blood, and clinical data. 
3.11.3 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 sequencing data will remain at GenomeDK (see 3.12.1.5), but the responsibility is transferred 
from the IMPROVE-IT2 study to the “Colorectal Cancer biobank MOMA, AUH”. The “Colorectal Cancer 
biobank MOMA, AUH” has been approved by the Danish Data Protection Agency 1-16-02-27-10. The 
biological materials, clinical and sequencing 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 destroyed. The storage of tissue and data in the research biobank is done in accordance with the 
Danish Health Act and the Act on Processing of Personal Data. New projects based on the biobank will only 
be initiated after the pertinent permissions have been obtained from the relevant regulatory committees. 
3.12 Molecular analysis 
3.12.1 Detection and quantification of circulating tumor DNA 
cfDNA will be isolated from the longitudinally collected plasma samples and blood spots using standard 
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3.12.1.1 ctDNA detection by digital PCR 
As part of the IMPROVE study, DNA from the patients’ tumor and leucocytes has been whole exome 
sequenced. The IMPROVE-IT2 study relies on this data for ctDNA analysis. Provided that a suitable mutation 
can be identified based on the whole exome sequencing data, the presence of ctDNA in the patients plasma 
samples will be monitored using a digital droplet PCR (ddPCR) assay targeting the mutation. The Department 
of Molecular Medicine currently holds a collection of assays targeting more than 90 different mutations 
observed in CRC. Approximately 80% of colorectal tumors (patients) will have at least one of these mutations, 
and hence the plasma samples from these patients can be monitored by this approach.  
3.12.1.2 ctDNA detection by ultra-deep targeted sequencing 
In the cases where a ddPCR assay cannot be provided, detection and quantification of ctDNA will be done 
using an optimized version of our recently developed ultra-sensitive targeted duplex sequencing approach54. 
Using this approach, we will sequence more than 5,000 unique DNA molecules from each plasma sample. 
Thereby, detection of somatic mutations with allele frequencies down to ~0.1% is possible, i.e. detection of 
one tumor molecule among 1,000 wild type DNA molecules in plasma. The sequencing will be done using 
Illumina sequencers. Our approach is much more sensitive than conventional sequencing. Conventional 
sequencing does not allow detection of mutations constituting less than 5% of the DNA in the analyzed 
sample. The poor sensitivity is due to a high error-rate of conventional sequencing. This makes it impossible 
to distinguish actual low-frequency mutations from errors. Our improved method includes multiple steps 
aimed at reducing the error rate. Including the use of unique strand-specific molecular barcodes (UMIs) and 
redundant sequencing of each cfDNA fragment. Thereby we can distinguish polymerase induced errors from 
true mutations. Errors will occur only in a subset of the redundant reads, while genuine mutations are present 
in all.  
Our ultra-deep targeted sequencing approach targets less than 2 million of the 3 billion bases in the human 
genome. The targets include the regions of the genome that are most frequently mutated in CRC (Table 1). 
Targeting these regions is sufficient to ensure that at least one somatic mutation will be identified in each 
CRC case and should therefore enable assessment of the ctDNA level in all included patients. The normal 
mucosa and CRC tumor tissue sequenced as part of the IMPROVE study serves as negative and positive 
references. 
 
Gene 
Regions sequenced 
TP53 
exons 5-8 (1792 bases) 
KRAS 
codons 12, 13, 61, 117, 146 (738 bases) 
SMAD2 
Selected frequently mutated regions (463 bases) 
SMAD4 
Selected frequently mutated regions (513 bases) 
FAM123B 
Selected frequently mutated regions (669 bases) 
SOX9 
Selected frequently mutated regions (693 bases) 
APC 
Selected frequently mutated regions (5477 bases) 
TCF7L2 
Selected frequently mutated regions (920 bases) 

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NRAS 
codons 12, 13, 61 (405 bases) 
BRAF 
codon 600 and others nearby (200 bases) 
FBXW7 
Selected frequently mutated regions (868 bases) 
PIK3CA 
8 hotspot mutations (905 bases) 
Table 1. CRC driver genes targeted by the CRC cfDNA sequencing panel 
3.12.1.3 ctDNA detection by low pass whole genome sequencing  
With the aim to develop a new platform for ctDNA detection, we will perform low pass (1-2x coverage) whole 
genome sequencing on plasma cfDNA from all patients. The sequencing will be done using Illumina 
sequencers. The data will be analyzed for a range of cancer associated features, including the cfDNA 
fragmentation patterns, copy number analysis mitochondrial DNA fraction55. We will investigate if these 
features can be used to discern the blood samples with and without ctDNA. As the golden standard, for the 
assessment we will use the digital PCR and ultra-deep targeted sequencing data and the clinical outcome of 
the patients.  
3.12.1.4 Bioinformatic processing of sequencing data 
Primary processing of sequencing data is done using Illumina Consensus Assessment of Sequence and 
Variation (CASAVA) software (v1.8), sequences are aligned against the human reference genome using the 
Burrows-Wheeler Alignment tool (BWA). Aligned data are processed using GATK best practice. Variant calling 
from whole exome sequencing data is done using MUTECT2. Variant calling from ultra-deep targeted 
sequencing data is done using a customized version of the R-package “deepsnv”. Analysis of processed and 
aligned low-pass whole genome sequencing data is done using custom build tools. 
3.12.1.5 Storage of sequencing data 
All sequencing data are processed and stored at GenomeDK. GenomeDK is a national high-performance 
computing facility for bioinformatics and life sciences managed by Center for Genome Analysis and 
Personalized Medicine, located at Aarhus University, Denmark. GenomeDK is used by Aarhus University 
Hospital and the Central Denmark Region for processing and storing sequencing data produced in the relation 
to the treatment of patients. IMPROVE-IT2 use the same security infrastructure and procedures as the 
Central Denmark Region. For duration of the storage see section 3.8.3.  
3.12.2 Quantification of carcinoembryonic antigen (CEA)  
Despite being an unspecific and insensitive marker of CRC, the blood-based protein biomarker CEA has been 
approved by both European and US regulatory authorities for surveillance after treatment for CRC. In this 
study CEA will be profiled in parallel to ctDNA and the performance of the two markers will be compared. 
3.13 Collection of clinical Information 
To enable identification recruitment of patients for IMPROVE-IT2, patients who have consented to participate 
in the IMPROVE study will be monitored to see if they full fill the IMPROVE-IT2 inclusion criteria (See 3.5.1.1). 
To enable assessment for potential biases between the patients accepting to participate and those who don’t 
the following information: CPR  number, tumor UICC stage, tumor location, surgical center, age, and gender, 
of all patients asked to participate will be stored in the project database. Patients who consent to participate, 
further accept that a central element of the project involves comparison and correlation of circulating tumor 
DNA assessments (gathered from the blood samples) to treatment and clinical outcome. Therefore, 
participating patients accept that the principal investigator, sponsor, and representative of the sponsor have 
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records and health registries to complete the study. Specifically information about surgical intervention, 
pathology reports, oncological intervention, and radiological evaluations aiming at detecting disease 
recurrence or assessing changes in tumor burden (e.g. during surveillance and treatment) will be collected. 
Data will be collected for up to ten years after the last protocolled patient visit.  
3.14 Definitions of predictor variables, co-variates and end-points 
3.14.1 Primary endpoint  
• 
Fraction of patients with relapse receiving intended curative resection or local treatment aiming at 
complete tumor destruction as defined by the Endpoint committee (3.14.4), within 3 years after 
surgery.  
3.14.2 Secondary endpoints  
• 
Time to clinical recurrence (TTCR) 
• 
Overall survival at 3 and 5 years (3-year OS and 5-year OS) 
• 
Time to molecular recurrence (TTMR) by either ctDNA or CEA 
• 
Clinical recurrence rate at time-intervals: randomization-12 months, 13-24 months, and 25-36 
months.   
• 
Quality of Life (QoL) by use of EORTC QLQ-C30, version 3.0, using global health status and functional 
scales only (17 items) 
• 
Fear of Cancer Recurrence Inventory (FCRI) (42 items) 
• 
Impact of Events Scale Cancer (IES-C) (15 items) 
• 
European Quality of life – 5 Dimensions (EQ-5D-5L) (5 items) 
• 
Adherence rate (AR) 
• 
Cost-effectiveness (CE) 
3.14.3 Safety Endpoints 
• 
Frequency and severity of adverse events (AE) in relation to the per protocol blood draws.  
• 
Frequency of allergic reaction to contrast material in relation to PET/CT imaging 
3.14.4 Definition of endpoints 
• 
Definition of curative or palliative intended treatment: An endpoint committee appointed by the trial 
office will prospectively assess the local site multidisciplinary team (MDT) notes and medical records 
of all relapse patients (both arms) and decide if the planned treatment is curative intended or 
palliative. The decision will not be provided to the local sites or otherwise influence the actual 
treatment delivered.  
• 
Overall survival from time of surgery is the outcome of primary interest and is calculated as the 
time from surgery to death from any cause. Due to immortal time from time of surgery until time of 
randomization (up till 8 months) an additional analysis will be performed calculating overall survival 
from time of randomization to death from any cause. 
• 
Time to clinical recurrence is calculated from randomization until detection of loco-regional 
recurrence or distant metastases, or death from colorectal cancer.  
• 
 
• 
Time to molecular recurrence is calculated from randomization until detectable ctDNA with 
censoring at time of death or end of follow-up. 
• 
Clinical recurrence rate is defined as total number of clinical recurrences detected divided by the 
total follow-up time during the specific time-interval. 

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• 
Cumulative incidence function of clinical recurrence is calculated treating clinical recurrence as 
event and death as competing event with censoring at end of follow-up.   
• 
Quality of Life is assessed by the questionnaires: QLQ-C30 (Appendix D), fear of cancer recurrence 
inventory, FCRI (Appendix E), impact of events scale cancer, IES-C (Appendix F), European Quality of 
life – 5 Dimensions, EQ-5D-5L (Appendix G). 
• 
Adherence rate assessed by the proportion of patients adhering 100% to the protocol. Protocol 
adherence is for both arms defined as attendance to all planned diagnostic surveillance according 
to the protocol and no extra planned surveillance (diagnostic examinations for clinical indications 
allowed). 
• 
The cost-effectiveness analysis will be carried out from a health care perspective and the health 
outcome measure in the cost-effectiveness analysis will be the total quality adjusted life years per 
group. 
 
3.15 Statistical analysis 
The statistical analysis plan for IMPROVE-IT2 has been published on June 25th 2024 (DOI: 
10.1101/2024.06.25.24309390). 
3.15.1 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. 
3.15.2 Analysis of OS, TTCR, QoL and CE 
A missed blood sample will not automatically lead to exclusion from the trial. However, multiple missed 
samples with consequences for the analysis may lead to patient exclusion (see monitoring). 
Data will be analyzed as intention-to-treat. Kaplan-Meier estimates will be used for the estimation of median 
times to recurrence (clinical and molecular), disease or death and their confidence intervals, stratified 
according follow-up intensity. Endpoints will be assessed using the log-rank test or a Cox regression model, 
with time to event (clinical or molecular recurrence or death) as response variable and intensity of follow-up 
as a factor. 
Quality of life and fear of recurrence analysis: As indicated in secondary objective 1 (Fejl! Henvisningskilde 
ikke fundet.) we will assess the  quality of life (QoL), fear of cancer recurrence (FCR), stress and anxiety of 
patients in the experimental and control arms. It is our hypothesis that there are no differences between the 
arms. The hypothesis will be assessed thrice during surveillance, at 12, 18 and 24 months, and after end of 
surveillance i.e. after the last scan and before month 38. The during surveillance assessments are done at 
two expected stress peaks (12 and 24 months - before a surveillance examination) and at an expected stress 
minimum (18 months – between two surveillance examinations).   
For measurement of QoL, FCR, stress and anxiety we use the validated Danish versions of the European 
Organization for Research and Treatment of Cancer QLQ-C30 health-related QoL questionnaire53, the Fear of 
Cancer Recurrence Inventory (FCRI)56, the Impact of Events Scale Cancer (IES-C)57, European Quality of life – 
5 Dimensions (EQ-5D-5L)58,59. 

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To have a baseline assessment of QoL, FCR, stress and anxiety all patients are asked to complete the 
questionnaires after completing adjuvant chemotherapy and before being informed of their randomization 
allocation and before starting surveillance.  
All assessments of differences between the two arms will be relative to the baseline assessment. 
We will also conduct an exploratory study of QoL, FCR, stress and anxiety of the patients in experimental arm 
that become ctDNA-positive and therefore receive high intensity PET-CT surveillance to identify the 
recurrence site (Secondary objective 7). These patients will be asked to complete the QoL questionnaires 
before each PET-CT. Changes in QoL, FCR, stress and anxiety will be longitudinally assessed (relative to 
baseline) from first PET-CT scan and until radiological recurrence. 
QoL data collection will be monitored closely in order to minimize missing forms. In case of non-random 
missing forms and/or values (items), imputation of data will be handled according to EORTC’s 
recommendations. 
Cost-effectivenes analysis: QoL/Utility weights for the quality adjusted life years parameter will be EQ-5D-
5L58–60 and QLU-C10D based on EORTC QLQ-C30 for sensitivity analysis. As we shall apply a lifetime horizon 
for the analysis, parametric survival functions will be used to extrapolate disease-free and overall survival 
curves beyond five years. Central cost drivers are i) costs of blood tests and the ctDNA analyses, ii) CT scans 
and PET-CT scans, and iii) differential costs of recurrence treatments due to early versus late discovery. The 
cost of the ctDNA detection methods will be estimated by bottom up cost calculations, including costs of 
material, lab use, and personnel time.  Depreciation of required equipment will also be performed and 
included to establish the cost price of ctDNA measurements. Medical resource use items, such as the costs 
of radiological imaging (PET-CT and CT scanning), treatment of recurrences (incl. surgery, radiofrequency 
ablation, chemotherapy), hospitalization, and non-molecular laboratory testing, will be extracted from the 
National Patient Register and if needed hospital records.   
Two approaches to costing will be applied and compared. First, total hospital costs for all study participants 
will be calculated from the National Patient Register. To avoid bias from extreme high costs unrelated to the 
intervention, such costs will be defined as being 10x higher than median hospital cost. If patients with such 
costs are present in the study, a panel of medical experts will be consulted to agree whether or not the costs 
are unrelated to the intervention, and, if found unrelated, the patients’ costs will then be excluded from the 
analysis. The second approach will calculate only cancer-related costs, as defined by having a cancer-
associated ICD10 code in the primary diagnosis in the National Patient Register.  Resource use will be linked 
to Danish tariffs. We will translate the cost-effectiveness assessments into business plans for hospitals and 
public finances to provide decision information for clinical and other decision makers. We shall also estimate 
productivity costs for labour market active patients but these are not included in the cost-effectiveness 
analysis.  
Costs are reported as follows: 1) costs in each arm in four groups: i) surveillance costs, and ii) recurrence 
costs (both conditional on recurrence and unconditionally for either surveillance programs), iii) other post-
surveillance health care costs, and iv) productivity costs;  2) in the cost-effectiveness analysis, i.e. in the ICER, 
cost components i-iii (but not iv) are added up; and 3) cost will be reported in intervals of one year i.e. 1st 
year costs, 2nd year costs, and 3rd year costs. All variables will be presented descriptively with point estimate 
and 95% confidence intervals. 
 To assess unintentional bias, the demographics and UICC stage of patients not willing to participate in the 
trial will be compared to the study population.   

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 Statistics will be performed in close collaboration with professional health research statisticians and cost-
effectiveness analysis will be performed by experts in health economics. 
3.15.3 Analysis of Safety 
AEs will be reported to the study coordinator. All AEs will be coded according to the standardized Medical 
Dictionary for Regulatory Activities (MedDRA). The coding will be performed by the study coordinator. 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 the medical records and from the Danish Colorectal Cancer Group 
database (DCCG database), Statistics Denmark (the BEF, BC, DREAM, FAIK, and CIV registries) and via 
questionnaires (e.g. QLQ-C30, FCRI, IES-C, and EQ-5D-5L). The information from the medical records will be 
entered into eCRF e.g. baseline information (including clinical TNM, WHO status), neoadjuvant treatment, 
pathology data, date of visits, adjuvant treatment, and radiological control 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 to pre-defined instructions. The QC will be performed before data is 
declared clean. 
4.2 Data Quality Assurance 
4.2.1 Case Report Forms (eCRFs) 
The Monitors will review and verify the data collected in the eCRFs against the source documents during 
monitoring visits. The Monitors will address the discrepancies found in the data and will ensure that 
corrections to the data are properly made and documented by the site personnel. All corrections will be 
documented in an audit track. 
4.2.2 PET/CT Second opinion board 
A second opinion board will be formed by the participating nuclear medicine and radiology departments and  
consist of experts in gastrointestinal imaging. The task of the board is to review negative PET-CT scans for 
quality and content. The local site PI is obligated to make arrangements to ensure that the local PET-CT center 
provides the scans and the description to the board immediately following the initial description. The board 
is obligated to perform its second opinion review within 5 working days after having received the scans and 
forward the conclusion to the local site PI. If the quality of the scan does not comply with protocol standard 
(3.10), a new PET-CT scan must be provided by the local PET-CT center immediately. When the quality of the 
PET-CT scan is acceptable the second opinion board will provide their diagnostic conclusion; the local PI must 
record the second opinion conclusion in RedCap.    
4.2.3 Monitoring 
The trial site will be visited by the Clinical Trial Manager (Monitor) periodically at times agreed with the 
Investigator. 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 conforms to applicable regulatory requirements and established rules for 
Good Clinical Practice (GCP).  

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Preferably at the time of each monitoring visit, the Monitor will 
• 
review the completed eCRFs to ascertain that items have been completed and that the data provided 
are accurate and obtained in the manner specified in the protocol. 
• 
verify that the data in the eCRF and DCCG database are consistent with the clinical records or other 
relevant records (Source Data Verification) and that trial results are recorded completely and 
correctly. 
• 
review the completeness of longitudinal blood sampling. In case of missing samples, the decision of 
letting the patient continue in the trial or be excluded will be at the discretion of the reviewing 
monitor.  
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 Investigator and other relevant 
personnel should be available during the monitoring visit and should devote sufficient time. 
The Monitor will continuously monitor the trial database and report on participant retention and 
completeness of follow-up collection. If problems or deviations are observed the monitor will inform the 
clinical coordinating investigator. Together they will formulate strategies to solve the problems. If necessary 
in collaboration with the steering committee. 
Data collection on QoL will be monitored closely in order to minimize missing forms. 
4.2.4 Training of staff 
All Investigators and staff carrying out observations of primary or other major efficacy variables involved in 
the trial should provide a curriculum vitae. The Investigator will keep a list of all personnel involved in the 
trial together with their function and trial related duties delegated. He/she will ensure that appropriate trial 
related training is given to all of the 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. 
4.2.5 Audit and inspections 
The trial site may be submitted to quality assurance audited by the Sponsor or someone appointed for this 
task by the Sponsor. The procedures of such a visit would be similar to those of a monitoring visit, and data 
already checked by the Monitor may be checked again. The Investigator and other relevant personnel must 
be available during the audit/inspection and must devote sufficient time. 
4.2.6 Changes in the approved Trial Protocol 
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 Authority for approval. 
5 Processing of personal data in the IMPROVE-IT2 study  
This study complies with the Data Protection Act and the General Data Protection Regulation (GDPR). 
The Health Act and the Helsinki II declaration are observed and will be complied with unconditionally. The 
project have been reported to the Central Denmark Region and is indexed in their internal record of ongoing 
research projects. 

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Throughout the study, all clinical data and samples will be labeled with non-personal identifiers. Information 
to identify individual patients will only be available to the study coordinator Prof. Claus Lindbjerg Andersen 
at the Department of Molecular Medicine (MOMA) and will only be used when collecting clinical information 
from the patient files or when a result is obtained indicating that a patient is qualified for randomization to 
receive higher follow-up intensity. 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. 
After the study has been completed and in relation to publication of the study results, the study data (health 
data and genomic data) will be transferred to the European restricted access database ”European Genome-
Phenome Archive”(https://ega-archive.org/) in a non-personalized format (pseudonymized). This is done to 
enable sharing of the data for confirmation and future research purposes and is put forward by the bodies 
funding the study and the scientific journals in which the results will be published. Data sharing is controlled 
and only researchers, who through their institutions accept to comply with the European data protection 
regulations and who after application has been approved by the IMPROVE-IT2 steering committee, will be 
granted access.  
6 Ethics and risk  
6.1 Ethics committee approval 
IMPROVE-IT2 has been approved by the Ethics Committee of the Central Denmark Region (j. no. 1-10-72-
162-19) 
6.2 Blood sampling  
Patients will have blood samples drawn as part of this project. Per draw 80 ml of blood will be collected by 
syringe and ctDNA positive patients will in addition be asked to donate 50-400 uL blood via a blood spot. This 
volume is clinically irrelevant (<2% of the total blood volume). Blood is sampled from a vein in the arm or by 
finger prick for blood spots, and this can be associated with temporary discomfort. The needle can cause 
local pain, bruising, and temporary redness at the injection site, but overall blood sampling is associated with 
minimal risk for the patients. In order to limit the inconvenience as much as possible the samples will be 
drawn as part of the routine management, whenever this is possible. Taken together, the benefits of 
participating in the study clearly outweigh the risk associated with the extra blood sampling.  
6.3 
Genomic sequence analysis 
In this study, we will perform ultra-deep targeted sequencing (~5000x coverage) of plasma DNA and 
leukocyte DNA to identify tumor specific alterations in the plasma DNA. We will disregard all similarities 
between the two samples i.e. all inherited mutations. Consequently, the risk of incidental finding a potential 
clinically relevant, genomic variant related to inherited diseases is extremely low, and practically 
hypothetical. We will also perform low pass whole genome sequencing (<2x coverage). This coverage is too 
low to allow identification of mutations. Instead, the data will be used to derive a range of cancer associated 
features such as cfDNA copy numbers, DNA fragmentation patterns, and the mitochondrial DNA fraction. 
These features we will be used in our assessment of the likelihood that the plasma contains tumor DNA.   
If genetic variants with potential clinical relevance are identified in the process, their importance will be 
evaluated by an expert committee, appointed by Department of Molecular Medicine. 
Patients are informed that they can decide to deny to receive information about incidental findings. If they 
do so, the decision must be indicated at the appropriate place on the consent form. 

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The committee is appointed when needed and the members will be chosen according to the potential 
disease. The committee will include a molecular biologist, specialized in genetic sequencing, and 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 penetrance) 
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 or not the patient (and/or his family) should be 
informed (by written letter) that the research accidentally has resulted in a finding, with potential influence 
on his or hers health, and that further information and advice on the matter is offered to him/her and/or 
potentially affected family members. If accepted, this will be initiated. 
Whether or not to provide feedback to relatives of deceased study participants or to study participants who, 
themselves, deny information about genetic issues, will be decided based on a medical perspective according 
to “DNVKs retningslinjer af 29. april 2013, sundhedslovens § 43, stk. 2, nr.2” and in “Autorisationsreglerne 
om lægers omhu og samvittighedsfuldhed”. 
6.4 
18FDG-PET/CT scan 
Patients becoming ctDNA positive in the experimental arm receive intensified follow-up with 18FDG-PET 
combined with a diagnostic contrast-enhanced CT-scan, 18FDG-PET/CT.  
Intravenous access for injection of 18FDG and CT-contrast is made under aseptic condition and may be 
associated with minor pain. Risk of infection at the puncture site is small.  
CT-contrast may cause a warm body sensation or a taste of metal in the mouth. Allergic reactions such as a 
rash or asthmatic symptoms occur in a few percent of patients. The CT-contrast is similar to the contrast used 
in the current follow-up after treatment for colorectal cancer. 
The total radiation dose of one 18FDG/CT is app. 23.6 mSv (18FDG 6 mSv and CT-scan x17.6 mSv). The exposure 
to a ctDNA positive patient who receive the maximum number of eight 18FDG/CT is 188.8 mSv. In comparison, 
the background radiation in Denmark is 3 mSv per year. The radiation dose of the 18FDG/CT does not reach a 
level causing risk of deterministic damages. There is no threshold dose below which it is certain that a 
stochastic effect cannot occur. Ionizing radiation is a known carcinogenic and increases the risk of cancer. 
The life-time risk of death of cancer in the general population is 25 %. The increased life-time risk of cancer 
in an adult from the background population exposed to a radiation dose similar to the maximum number of 
18FDG/CT in this protocol is less than 3 %. The increased risk of a radiation-induced secondary cancer in the 
population included in IMPROVE-IT2 is considerably less (<1%), as mean age at time of CRC is about 70 years 
and secondary cancer risk decreases substantially with increasing age of exposure. In contrast, the ctDNA 
positive patients among stage III CRC patients have an exceedingly high relapse risk approaching 100 %. The 
overall 5-year survival is only 9 % among relapsing patients. This is due to relapse discovered at an advanced 
stage with curative intended treatment in only 15 %. The potential of intensified follow-up in ctDNA positive 
patients identifying relapse, at an early stage, where curative treatment is still possible, clearly outweigh the 
risk of a radiation-induced secondary cancer. 

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7 Publication and reporting from the trial 
The results of the IMPROVE-IT2 study 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 published as 
soon as at it is considered professionally sound. Authorship will follow the Vancouver recommendations. If 
results unexpectedly cannot be published in journals other means on publication will be sought such via the 
partners´ institutional websites.  
8 Economy 
IMPROVE-IT2 is a researcher initiated study. The study (incl. experimental costs and salaries) is supported by 
research grants given to Claus Lindbjerg Andersen (3,900,000 DKK from Danish Cancer Society and 
12,500,000 DKK from Innovation Fund Denmark). The research grants are paid to and administered by Aarhus 
University and Aarhus University Hospital. The investigators have no financial relation to the grant provider 
nor any financial interests in the project. The sponsor, the clinical coordinating investigator and the site 
investigators have no competing interests. 
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). 

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