Adjuvant Chemotherapy for Prevention of Recurrence in Patients With Detectable ctDNA After Surgery in High-Risk Rectal Cancer
Phase: PHASE3
Conditions: Rectal Cancer
Sponsor: Erasmus Medical Center

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REACT 
 
Adjuvant chemotherapy for prevention of recurrence in patients with  
detectable ctDNA after surgery in high-risk rectal cancer. 
A phase III, multicentre study.  
 
 
 
 
 
 
 
 

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CLINICAL TRIAL TITLE  
‘Adjuvant chemotherapy for prevention of recurrence in patients with detectable ctDNA after surgery 
in high-risk rectal cancer.’ 
Protocol ID 
NL82006.078.22 
Short title 
REACT 
EU trial number  
2024-517700-12-00 
Protocol version 
4.0 
Protocol date 
18-06-2025 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
 
Erasmus MC, University Medical Center Rotterdam 
Department of Surgery 
Dr. Molewaterplein 40 
3015 GD Rotterdam, The Netherlands 
Legal representative(s) of the 
sponsor: 
Erasmus MC, University Medical Center Rotterdam 
Prof. dr. J.M. Hendriks 
Head of Department of Surgery 
Dr. Molewaterplein 40 
3015 GD Rotterdam, The Netherlands 
Principal investigator (in Dutch: 
hoofdonderzoeker/uitvoerder) 
 
 
 
 
 
Co-investigator 
 
 
 
 
 
 
Multicenter research: per site 
Erasmus MC, University Medical Center Rotterdam 
Prof. dr. C. Verhoef  
Department of Surgery 
Rotterdam, The Netherlands 
Phone: 0031 10 704 1902 
E-mail: c.verhoef@erasmusmc.nl 
 
Erasmus MC, University Medical Center Rotterdam 
Prof. dr. H.M.W. Verheul 
Department of Medical Oncology 
Rotterdam, The Netherlands 
Phone: 0031 10703 0776 
E-mail: h.verheul@erasmusmc.nl  
 
Reinier de Graaf Hospital 
Dr. A. Verschoor 
Department of Medical Oncology 
Delft, The Netherlands 
Phone: 015 2604080 
E-mail: a.verschoor@rdgg.nl 
 
Bravis Hospital 
Dr. S. Boudewijns 
Department of Medical Oncology 
Roosendaal, The Netherlands 
Phone: 088 7066820 
E-mail: s.boudewijns@bravis.nl 
 

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Franciscus Gasthuis 
Dr. M. Verseveld 
Department of Surgical Oncology 
Rotterdam, The Netherlands 
Phone: 010 4616163 
E-mail: m.verseveld@franciscus.nl 
 
Albert Schweitzer Hospital 
Dr. E. Belt 
Department of Surgical Oncology 
Dordrecht, The Netherlands 
Phone: 078 65232350 
E-mail: e.j.t.belt@asz.nl 
 
ZorgSaam Hospital 
Dr. M.P.S. Sie 
Department of Medical Oncology 
Terneuzen, The Netherlands 
Phone: 0115 688202 
E-mail: m.sie@zzv.nl  
 
Ikazia Hospital 
Dr. J. Nonner 
Department of Surgical Oncology 
Rotterdam, The Netherlands 
Phone: 010 2975220 
E-mail: j.nonner@ikazia.nl 
 
Jeroen Bosch Hospital 
Dr. H. Schut 
Department of Medical Oncology 
‘s-Hertogenbosch, The Netherlands 
Phone: 073 5538225 
E-mail: h.schut@jbz.nl 
 
Elisabeth Tweesteden Hospital 
Dr. L. Beerepoot 
Department of Medical Oncology 
Tilburg, The Netherlands 
Phone: 013 2213069 
E-mail: l.beerepoot@etz.nl 
 
Maasstad Hospital 
Dr. B. Haberkorn 
Department of Medical Oncology 
Rotterdam, The Netherlands 
Phone: 010 2911840 

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E-mail: haberkornb@maasstad.nl 
 
Admiraal de Ruyter Hospital 
Dr. H. van Halteren 
Department of Medical Oncology 
Goes, The Netherlands 
Phone: 088 1254510 
E-mail: hk.vanhalteren@adrz.nl 
 
 
Coordinating investigator/project 
leader 
Erasmus MC, University Medical Center Rotterdam 
Prof. Dr. C. Verhoef  
Department of Surgery 
Rotterdam, The Netherlands 
Phone: 0031 10 704 1902 
E-mail: c.verhoef@erasmusmc.nl 
Study coordinators  
Erasmus MC, University Medical Center Rotterdam  
Drs. M. Ubink, MD 
Department of Surgery 
Rotterdam, The Netherlands 
E-mail: m.ubink@erasmusmc.nl  
 
Erasmus MC, University Medical Center Rotterdam  
Drs. D.M. Mens, MD 
Department of Surgery 
Rotterdam, The Netherlands 
E-mail: d.mens@erasmusmc.nl  
Funding party 
Erasmus MC Foundation 
Independent expert 
Erasmus MC, University Medical Center Rotterdam  
Dr. J.L. Boormans 
Department of Urological Oncology 
Rotterdam, The Netherlands 
Tel. 0031 (0)107030538 
Laboratory sites  
Erasmus MC, University Medical Center Rotterdam 
Medical Oncology Laboratory 
Dr. Molewaterplein 40 
3015 GD Rotterdam 
 
 
Pharmacy  
 
Not applicable 
 
  

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PROTOCOL SIGNATURE SHEET 
Name 
Signature 
Date 
Sponsor or Legal representative: 
Prof. dr. J.M. Hendriks, Vascular Surgeon 
Erasmus MC, University Medical Center 
Rotterdam 
 
Head of Department: 
Prof. dr. J.M. Hendriks, Vascular Surgeon 
Erasmus MC, University Medical Center 
Rotterdam 
 
 
Coordinating Investigator: 
Prof. dr. C. Verhoef, Surgeon 
Erasmus MC, University Medical Center 
Rotterdam 
 
Co-investigator: 
Prof. dr. H.M.W. Verheul, Medical Oncologist 
Erasmus MC, University Medical Center 
Rotterdam 
 
 
Principal Investigators per site: 
 
Dr. A. Verschoor, Medical Oncologist 
Reinier de Graaf Hospital 
Delft 
 
Dr. S. Boudewijns, Medical Oncologist 
Bravis Hospital 
Roosendaal 
 
Dr. M. Verseveld, Surgeon 
Franciscus Gasthuis Hospital 
Rotterdam 
 
Dr. E. Belt, Surgeon 
Albert Schweitzer Hospital 
Dordrecht 
 
Dr. M.P.S. Sie, Medical Oncologist 
ZorgSaam Hospital 
Terneuzen 
 
Dr. J. Nonner, Surgeon 
Ikazia Hospital 
Rotterdam 
 
Dr. H. Schut, Medical Oncologist 
 
 

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Jeroen Bosch Hospital 
‘s-Hertogenbosch 
 
Dr. L. Beerepoot, Medical Oncologist 
Elisabeth Tweesteden Hospital 
Tilburg 
 
Dr. B. Haberkorn, Medical Oncologist 
Maasstad Hospital 
Rotterdam 
 
Dr. H. van Halteren, Medical Oncologist 
Admiraal de Ruyter Hospital 
Goes 
 
 
 
 
 

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DOCUMENT HISTORY 
Document 
Date of version 
Summary of Changes 
Protocol version no. 4.0 
 
06-03-2025 
- 
Addition of 10 centres for multicenter 
study 
- 
Added the results of the PRODIGE-23 
study in the background information 
- 
Updated preliminary research 
- 
Added additional ctDNA at first follow-up 
and at progression as secondary 
endpoints 
- 
Described overall study duration and 
follow-up 
- 
Described the patient participation in the 
design of the trial 
- 
Updated the in- and exclusion criteria 
- 
Described the considerations of in-vitro 
diagnostics 
- 
Described the lifestyle restrictions 
- 
Described the screening procedure 
- 
Updated the ctDNA analysis procedure 
- 
Elaborated on the data collection 
procedure 
- 
Described the efficacy and safety 
assessments 
- 
Updated the safety reporting according 
to the CTR regulations 
- 
Described the analysis sets 
- 
Elaborated on the randomisation 
procedure (stratification and block sizes) 
- 
Described potential reasons for early 
termination of the trial 
- 
Described procedures for accounting for 
missing data and potential deviations 
from statistical plan 
- 
Elaborated on the monitoring plan 
- 
Described the collection and storage of 
biological samples 
Original protocol 
 
10-07-2024 
Not applicable 
 
 
CONFIDENTIALITY STATEMENT 
This document contains confidential information that must not be disclosed to anyone other than 
the sponsor, the investigative team, regulatory authorities, and members of the Research Ethics 
Committee. 
 
 

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TABLE OF CONTENTS 
 
1. 
ABBREVIATIONS ............................................................................................................................. 12 
2. 
SYNOPSIS ....................................................................................................................................... 14 
3. 
INTRODUCTION AND RATIONALE .................................................................................................. 16 
3.1 
Therapeutic condition and current treatment status .....................................................16 
3.2 
Clinical trial rationale ................................................................................................17 
3.3 
Mechanism of action, Drug class.................................................................................20 
3.4 
Rationale for Dose Regimen/Dose Justification ............................................................20 
4. 
STRUCTURED RISK ANALYSIS ......................................................................................................... 20 
4.1 
Potential issues of concern ........................................................................................20 
4.1.1 
Level of knowledge about mechanism of action ........................................................... 20 
4.1.2 
Previous exposure of human beings ............................................................................. 20 
4.1.3 
Induction of the mechanism in animals and/or ex-vivo ................................................ 20 
4.1.4 
Selectivity of the mechanism ........................................................................................ 20 
4.1.5 
Analysis of potential effect ............................................................................................ 20 
4.1.6 
Pharmacokinetic considerations ................................................................................... 20 
4.1.7 
Predictability of effect ................................................................................................... 21 
4.1.8 
Interaction with other products .................................................................................... 21 
4.1.9 
Managing of effects ....................................................................................................... 21 
4.1.10 
Study population ........................................................................................................... 21 
4.2 
Overall synthesis of the direct risks for the research subjects .........................................21 
5. 
OBJECTIVES AND ENDPOINTS ........................................................................................................ 22 
6. 
STUDY PLAN AND DESIGN ............................................................................................................. 24 
6.1 
Trial Design ..............................................................................................................24 
6.2 
Number of Patients ...................................................................................................25 
6.3 
Overall study duration and follow-up ..........................................................................25 
6.4 
Patient participation .................................................................................................25 
7. 
STUDY POPULATION ...................................................................................................................... 26 
7.1 
Population ...............................................................................................................26 
7.2 
Inclusion criteria .......................................................................................................26 
7.3 
Exclusion criteria ......................................................................................................26 
7.4 
Vulnerable populations and clinical trials in emergency situations ..................................26 
8. 
STUDY TREATMENTS ..................................................................................................................... 27 
8.1 
Investigational Medicinal Product(s) (IMP(s)) ...............................................................27 
8.1.1 
Name and description of the IMP ................................................................................. 27 
8.1.2 
Status of development of the IMP ................................................................................ 27 
8.1.3 
Description and justification of dosage and route of administration ........................... 27 

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8.2 
Comparator IMP(s) ...................................................................................................27 
8.3 
Placebo ...................................................................................................................28 
8.4 
Auxiliary Medicinal Product(s) (AxMP(s)) .....................................................................28 
8.4.1 
Name and description of the AxMP .............................................................................. 28 
8.4.2 
Statement on authorisation and justification unauthorised AxMP (if applicable) ....... 28 
8.4.3 
Description and justification of dosage and route of administration ........................... 28 
8.5 
Additional considerations for trials involving a medical device .......................................28 
8.6 
Additional considerations for trials involving an in-vitro diagnostic or companion diagnostic
 
28 
8.7 
Preparation and labelling of the study treatment(s) ......................................................28 
9. 
OTHER TREATMENTS AND RESTRICTIONS..................................................................................... 29 
9.1 
Concomitant therapy ................................................................................................29 
9.1.1 
Permitted medication(s) ................................................................................................ 29 
9.1.2 
Prohibited medication(s) ............................................................................................... 29 
9.2 
Lifestyle restrictions ..................................................................................................29 
9.2.1 
Contraception measures ............................................................................................... 29 
9.2.2 
Other requirements ....................................................................................................... 29 
10. 
TRACEABLILITY, STORAGE, ACCOUNTABLILITY AND COMPLIANCE ........................................... 29 
10.1 
Traceability and storage of the study treatment(s)? ......................................................29 
10.2 
Accountability of the study treatment(s) and compliance ..............................................29 
11. 
STUDY ASSESSMENTS AND PROCEDURES ................................................................................. 30 
11.1 
Screening procedure .................................................................................................30 
11.2 
Randomisation, blinding and treatment allocation........................................................30 
11.3 
Study procedures and assessments .............................................................................30 
11.3.1 
Efficacy assessments ..................................................................................................... 34 
11.3.2 
Safety assessments ........................................................................................................ 34 
12. 
STUDY DISCONTINUATION AND COMPLETION ......................................................................... 35 
12.1 
Definition End of Trial................................................................................................35 
12.2 
Criteria for temporary halt and early termination of the clinical trial ...............................35 
12.3 
Discontinuation/withdrawal of individual subjects ........................................................35 
12.4 
Arrangements for subjects after their participation in the clinical trial ended ..................35 
13. 
SAFETY REPORTING ................................................................................................................... 36 
13.1 
Definitions ...............................................................................................................36 
13.1.1 
Adverse events (AEs) ..................................................................................................... 36 
13.1.2 
Serious adverse events (SAEs) ....................................................................................... 36 
13.1.3 
Suspected unexpected serious adverse reactions (SUSARs) ......................................... 36 
13.2 
Recording of AEs/SAEs/SUSARS ..................................................................................36 
13.3 
Reporting of AEs and SAEs .........................................................................................37 
13.3.1 
Reporting of SAEs by the investigator to the sponsor ................................................... 37 

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13.3.2 
List of SAEs which do not require immediate reporting and procedure for reporting . 37 
13.4 
Follow-up of adverse events ......................................................................................37 
13.5 
Reporting of SUSARs by the sponsor to EudraVigilance .................................................37 
13.6 
Annual safety report .................................................................................................37 
13.7 
Unblinding procedures for safety reporting .................................................................38 
13.8 
Temporary halt for reasons of subject safety ...............................................................38 
13.9 
Urgent safety measures and other relevant safety reporting .........................................38 
13.10 
Data Safety Monitoring Board (DSMB)/Data Monitoring Committee (DMC) .................38 
14. 
STATISTICAL ANALYSIS ............................................................................................................... 39 
14.1 
Description of statistical methods ...............................................................................39 
14.2 
Analysis sets .............................................................................................................39 
14.3 
Participant demographics and other baseline characteristics .........................................39 
14.4 
Randomisation and blinding .......................................................................................39 
14.5 
Sample size, trail power and level of significance used ..................................................39 
14.6 
Planned analysis .......................................................................................................41 
14.6.1 
Analysis primary endpoint ............................................................................................. 41 
14.6.2 
Analysis secondary endpoint(s) ..................................................................................... 41 
14.6.3 
Analysis other study parameters/endpoints ................................................................. 41 
14.7 
Interim analysis ........................................................................................................42 
14.8 
(Statistical) criteria for termination of the trial .............................................................42 
14.9 
Procedure for accounting for missing, unused and spurious data ...................................42 
14.10 
Procedure for reporting any deviation(s) from the original statistical plan ...................42 
15. 
ETHICAL CONSIDERATIONS ....................................................................................................... 42 
15.1 
Declaration of Helsinki ..............................................................................................42 
15.2 
Recruitment and informed consent procedures ...........................................................42 
15.3 
Benefits and risks assessment, group relatedness .........................................................44 
15.4 
Compensation for injury ............................................................................................44 
15.5 
Compensation for subjects ........................................................................................44 
15.6 
Compensation for investigators ..................................................................................44 
15.7 
Other ethical considerations ......................................................................................44 
16. 
ADMINISTRATIVE ASPECTS, MONITORING AND CONFIDENTIALITY ......................................... 44 
16.1 
Approval initial application and substantial modifications .............................................44 
16.2 
Monitoring ...............................................................................................................45 
16.3 
Recording, handling and storage of information ...........................................................45 
16.3.1 
Handling of data and data protection ........................................................................... 45 
16.3.2 
Source documents and case report forms (CRF) ........................................................... 45 
16.3.3 
Clinical trial master file and data archiving ................................................................... 46 
16.3.4 
Collection and storage of biological samples ................................................................ 46 
16.4 
Audits and inspections and direct access to source data/documents ..............................46 
16.5 
Reporting of serious breaches ....................................................................................46 
16.6 
Notification of the start and the end of the recruitment ................................................46 

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16.7 
Temporary halt/(early) termination ............................................................................47 
16.7.1 
Temporary halt/early termination for reasons not affecting the benefit-risk balance 47 
16.7.2 
Temporary halt/early termination for reasons of subject safety .................................. 47 
16.8 
Summary of the results .............................................................................................47 
16.9 
Public disclosure and publication policy .......................................................................47 
17. 
REFERENCES .............................................................................................................................. 49 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. ABBREVIATIONS  
 
ABR 
ABR form, General Assessment and Registration form, is the application form that is 
required for submission to the accredited Ethics Committee (In Dutch, ABR = Alge-
mene Beoordeling en Registratie) 
AE 
Adverse Event 
AR 
Adverse Reaction 
AxMP 
Auxiliary Medicinal Product 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: Centrale Com-
missie Mensgebonden Onderzoek 
CtDNA 
Circulating tumour DNA 
CfDNA 
Cell-free DNA 
CHIP 
Clonal haematopoiesis of indeterminate potential 
CRO 
Contact Research Organisation 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
e-CRF 
Electronic Case Report Form 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IKNL 
Integraal Kankercentrum Nederland 
IMP 
Investigational Medicinal Product 
IMPD 
Investigational Medicinal Product Dossier 
METC  
Medical research ethics committee (MREC); in Dutch: medisch ethische toetsing 
commissie (METC) 
MS 
Member State 
NCR 
Netherlands Cancer Registry 
NGS 
Next generation sequencing 

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PCR 
Polymerase chain reaction 
PI 
Principal Investigator 
PLCRC 
Prospective Dutch ColoRectal Cancer 
PROMs 
Patient Reported Outcomes 
RSI 
Reference Safety Information 
SAE 
Serious Adverse Event  
SmPC 
Summary of Product Characteristics  
SOP 
Standard Operating Procedure 
Sponsor 
The sponsor is the party that commissions the organisation or performance of the 
research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A party that 
provides funding for a study but does not commission it is not regarded as the spon-
sor, but referred to as a subsidising party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TME 
Total mesorectal excision 
TwiCs 
Trial within cohorts 
UMIs 
unique molecule identifiers 
WBP 
Wet Basisregistratie Personen  
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; in Dutch: 
Uitvoeringswet AVG 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-weten-
schappelijk Onderzoek met Mensen 
 
 
 
 
 
 
 
 
 
 
 

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2. SYNOPSIS 
2024-517700-12-00 - ‘Adjuvant chemotherapy for prevention of recurrence in patients with 
detectable ctDNA after surgery in high-risk rectal cancer.’ 
 
Rationale 
Rectal cancer is a worldwide cause of cancer related mortality.(1) The incidence of rectal cancer in 
the Netherlands is approximately 3500 patients per year.(2) The introduction of combined neoadju-
vant (chemo)radiotherapy and total mesorectal excision (TME) has significantly reduced the local re-
currence rate, but distant recurrence rates remain around 30%.(3-5) Recurrences are likely to derive 
from residual local disease or subclinical metastatic disease (minimal residual disease). These micro 
metastases are undetectable by the currently used imaging techniques but still present after surgery. 
Adjuvant chemotherapy might be beneficial for patients at high risk for recurrence. However, there 
are only a few randomised controlled trials on perioperative chemotherapy available. Studies on ad-
juvant chemotherapy in rectal cancer yielded conflicting results.(6, 7) As a consequence, treatment 
with adjuvant chemotherapy in patients with rectal cancer is not evidence based and therefore not 
standard of care in the Netherlands. Recent studies suggest that preoperative intensive chemother-
apy with radiotherapy, compared to standard chemotherapy and radiotherapy, resulted in a pro-
longed disease-free survival (8, 9) and overall survival. (9) However, this was at the cost of increased 
toxicity, and whether the observed improvement in overall survival can be attributed to the addition 
of neo-adjuvant intensive chemotherapy is under debate.(10) Consequently, there is an urgent need 
for biomarkers to identify those patients at high risk to recur after standard treatment, to select the 
patients that might benefit the most from adjuvant chemotherapy. 
 
Objective 
To investigate disease-free survival in patients with high-risk rectal cancer by treating these patients 
with adjuvant chemotherapy in case of detectable ctDNA after surgery. 
 
Main trial endpoints  
The primary endpoint of the study will be disease-free survival in the intention-to-treat population, 
calculated from the date of surgery to the date of recurrence or death from any cause of the patient, 
whichever occurs first. 
 
Secondary trial endpoints  
Secondary outcomes will be disease-free survival, carried out as per protocol analysis to analyse pure 
treatment effect. In addition, overall survival will be calculated measured from the date of surgery to 
the date of death from any cause. Quality of life will be assessed in both groups by obtaining already 
completed questionnaires provided by the PLCRC cohort study to compare the effect of adjuvant 
chemotherapy on quality of life. The robustness of ctDNA as biomarker will be analysed by 
comparing the disease-free survival of patients with detectable ctDNA who are not treated adjuvant 
chemotherapy (control group) with patients with undetectable ctDNA. The clearance of ctDNA of the 
patients who received adjuvant chemotherapy in the experimental group will be compared with the 
patients in the control group. Lastly, the co-occurrence of ctDNA in peripheral blood at the timing of 
detection of recurrent disease on imaging will be studied. 
 
 
 
 

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Trial design  
The proposed study is conducted within the prospective Dutch ColoRectal Cancer (PLCRC) cohort and 
follows the trial within cohort (TwiCs) design, i.e. a randomised controlled trial within a prospective 
cohort.(11) 
 
Trial population  
Patients ≥ 18 years of age with primary resected rectal cancer that meet the inclusion criteria, 
participating in the PLCRC cohort with informed consent for randomisation and considered physically 
fit for adjuvant chemotherapy are eligible.   
 
Interventions 
Patients with detectable ctDNA after surgery and randomised to the experimental group will be 
offered adjuvant chemotherapy within 8 weeks of surgery and no longer than 12 weeks after 
surgery. Adjuvant chemotherapy consists of 6 cycles of 5FU/folinic acid and oxaliplatin (FOLFOX) 
every 2 weeks, or 4 cycles of capecitabine and oxaliplatin (CAPOX). Duration of treatment will be 3 
months (12 weeks). 
 
Ethical considerations relating to the clinical trial including the expected benefit to the individual 
subject or group of patients represented by the trial subjects as well as the nature and extent of 
burden and risks  
In current clinical practice there is no indication for adjuvant chemotherapy for patients after surgery 
for primary rectal cancer. Therefore all participating patients have no indication for adjuvant chemo-
therapy. Patients randomised to the experimental group will be offered adjuvant chemotherapy to 
reduce recurrence. According to routine clinical care, patients receiving adjuvant chemotherapy will 
undergo blood withdrawals and visit their treating physician before every cycle of chemotherapy. 
The combination chemotherapy schedule of CAPOX and FOLFOX is commonly administered in the 
adjuvant setting in current practice for colorectal cancer, therefore the risks and toxicity of the used 
adjuvant chemotherapy are well-known. The majority of side-effects are manageable and transient. 
The risk of the withdrawal of extra tubes of blood during regular blood withdrawal in all study partici-
pants is negligible. The benefit for participants enrolled in this trial is the potential chance to reduce 
their risk of recurrence with adjuvant chemotherapy. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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3. INTRODUCTION AND RATIONALE 
3.1 Therapeutic condition and current treatment status 
Rectal cancer is a worldwide cause of cancer related mortality.(1) The incidence in the Nether-
lands is approximately 3500 patients per year.(2) The introduction of combined neoadjuvant 
(chemo)radiotherapy and total mesorectal excision (TME) significantly reduced the recurrence 
rate, but recurrence rates remain high.(3-5) Approximately 30% of the patients treated with cura-
tive intent for rectal cancer will develop recurrent disease, locally and distant.(4, 12, 13) Recur-
rences are likely to derive from micro metastases (minimal residual disease). Minimal residual dis-
ease is undetectable by imaging techniques but still present after surgery. Adjuvant chemother-
apy can be beneficial for patients at high risk for recurrence by eliminating minimal residual dis-
ease.  
 
The evidence for adjuvant chemotherapy in rectal cancer is sparse and there is no international 
consensus. There are few randomised controlled trials available regarding the effect of adjuvant 
chemotherapy for rectal cancer. The EORTC-22921-(14), I-CNR-RT-(15), PROCTOR-SCRIPT-(16) and 
CHRONICLE-trial(17), showed no benefit of adjuvant chemotherapy on overall and disease-free 
survival, but the QUASAR-trial(18) did. Meta-analyses also presented contradicting results. A 
Cochrane review(19) displayed an overall beneficial effect of adjuvant chemotherapy on overall 
and disease-free survival, however meta-analyses by Bujko(6) and Breugom(7) demonstrated no 
improvement. International efforts to improve overall survival outcomes of patients with rectal 
cancer, for example by the use of more intensified neoadjuvant and adjuvant treatment, have so 
far been to no avail.(8, 20) Toxicity of systematic chemotherapy remains an important downside 
of implementing these strategies as standard of care.  
 
The recent years total neo-adjuvant treatment (TNT) has become of interest, which comprises  
the administration of both systemic chemotherapy and chemoradiotherapy before surgical  
resection. The Dutch RAPIDO study has investigated short course radiotherapy with subsequent 
doublet systematic chemotherapy (CAPOX/FOLFOX4) in comparison to standard treatment with 
chemoradiation in patients with locally advanced rectal cancer.(8) This study demonstrated a ben-
efit of the RAPIDO treatment protocol with a 3-year disease related treatment failure improve-
ment of 6.7%. However, the 3-year overall survival was similar between both groups and patients 
receiving the RAPIDO treatment protocol experienced more grade 3-4 adverse events (48% vs 
25%). The modest improvement of disease-free survival with the RAPIDO treatment regime im-
plies a significant number of patients being overtreated using an intensified treatment regimen, at 
the cost of increased toxicity without a certain overall survival benefit. 
 
The PRODIGE-23 trial, which compared neo-adjuvant treatment using mFOLFIRINOX followed  
by chemoradiotherapy to chemoradiotherapy alone in patients with LARC, did demonstrate an 
overall survival benefit favoring the group who received TNT.(9) However, the overall survival  
curve shows an early effect on overall survival around 4 months with more or less parallel lines  
from that moment on. Based on the early studies on the effect of chemotherapy in non-meta-
static disease, an effect of chemotherapy on overall survival is expected in the long term  
around 18 months.(21) Therefore, the observed overall survival benefit in the PRODIGE-23  
trial is unlikely the result of the studied intervention and must be explained by other causes. 
 
 

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Patient selection for (neo)adjuvant chemotherapy for rectal cancer remains a major clinical di-
lemma. In countries where adjuvant chemotherapy is standard of care, selection is currently 
based on TNM staging, overtreating numerous patients as a consequence. Biomarkers to identify 
patients at high risk to recur are urgently needed to offer a more tailored treatment regime, 
hereby reducing the chance of unnecessary toxicity and impaired quality of life. 
 
3.2 Clinical trial rationale 
Circulating tumour DNA (ctDNA) in peripheral blood samples is a potential biomarker to identify 
patients at high risk for recurrence as these patients may particularly benefit from adjuvant chem-
otherapy.(22-24) Identification of patients at high risk with ctDNA and treatment of these patients 
with adjuvant chemotherapy could lower their recurrence rate and improve disease-related over-
all survival. 
 
Circulating tumour DNA 
CtDNA is a promising biomarker that is considered to be an important diagnostic tool for the de-
tection of minimal residual disease.(22-24) CtDNA is part of the total amount of small fragments 
of DNA in the blood, called cell-free DNA. These fragments are shed into the bloodstream from 
dying cells during cellular turnover or other forms of cell death. The utility of ctDNA has already 
been suggested to aid in clinical decision-making in metastatic colorectal cancer. For example, re-
sistance to monoclonal anti-EGFR antibodies such as cetuximab and panitumumab due to EGFR 
and downstream KRAS mutations, can be detected by ctDNA analysis as early as 10 months be-
fore radiologic disease progression is observed.(25-28) The detection limit of ctDNA analysis 
methods to detect tumour DNA in the total amount of cell-free DNA is below 0.05%, while still 
preserving a specificity of >99.99%.(23, 29)  
 
The detection and use of ctDNA in patients with non-metastatic colorectal cancer have already 
been investigated. In colon cancer, the detection of ctDNA after surgery was strongly associated 
with recurrence of disease, with recurrence rates up to 79% within 2 years after surgery for pa-
tients with detectable ctDNA, compared to only 10% in patients without detectable ctDNA.(30) 
More importantly, although only shown in small sample sizes, the administration of adjuvant 
chemotherapy was able to change ctDNA from positive to negative.  
 
In a recently published study with 159 patients with locally advanced rectal cancer (LARC) ctDNA 
status stratified cases into very high and low risk for recurrence groups.(31) Prior to neoadjuvant 
(chemo)radiotherapy, 122 (77%) patients had detectable ctDNA. After surgery, 19 patients (12%) 
had detectable ctDNA of which 58% recurred during follow-up (median 24 months). In contrast, 
recurrence occurred in only 8.6% of the patients with a negative ctDNA status (hazard ratio 13, 
95%CI 5.5-31, p<0.001). The prognostic value of detectable ctDNA for recurrence was even 
stronger in patients with a high pathological stage (ypT3-4 and ypN1-2), demonstrated by recur-
rence rates up to 89% after 2 years. 
 
Khakoo et al. investigated the role of ctDNA in patients with LARC and similarly showed that all 3 
patients with detectable ctDNA after surgery had recurrent disease compared with none of the 20 
patients with undetectable ctDNA (P = 0.001).(32) Other recently published studies among pa-
tients with LARC show promising results regarding the prognostic value and the applicability of 
ctDNA for the prediction of treatment response after neoadjuvant chemoradiation.(33, 34) Fur-

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thermore, data from a recent trial in stage II colon cancer suggest that ctDNA positive patients ap-
pear to derive considerable benefit from adjuvant treatment in terms of recurrence-free survival, 
and that a ctDNA-guided treatment approach can reduce the number of patients who received 
adjuvant therapy whilst not altering the risk of recurrence.(35) 
 
Collectively, these studies have consistently shown the high potential of ctDNA as future bi-
omarker for detecting minimal residual disease after surgery. It can be concluded from these re-
sults that postoperative ctDNA analysis stratifies patients into very high and low risk groups for 
recurrence, independent of conventional clinicopathological risk factors. Unfortunately, the use of 
ctDNA is not yet implemented in daily practice. The potential benefit of adjuvant chemotherapy in 
patients with detectable ctDNA after surgery should be established. 
 
Note: a systematic review performed by the REACT research team, including a more comprehen-
sive overview of ctDNA studies in the field of rectal cancer, can be found in the REACT literature 
review. 
 
Rationale 
Use of ctDNA in clinical practice is not yet incorporated in clinical guidelines. Further research, val-
idation and standardization needs to be done to enable the clinical application of ctDNA for diag-
nosis and monitoring of cancer. ctDNA in peripheral blood samples is a potential biomarker to 
identify patients at high risk for recurrence after curative surgery for rectal cancer and these high 
risk patients will possibly benefit from adjuvant chemotherapy. 
 
The aim of this study is to reduce the recurrence rate and improve overall survival in patients with 
rectal cancer, by treating patients with adjuvant chemotherapy in case of detectable ctDNA after 
surgery. Our hypothesis is that adjuvant chemotherapy in high-risk rectal cancer patients with de-
tectable ctDNA after surgery will lead to approximately 25% lower recurrence rate within two 
years compared to the current standard of care in patients with detectable ctDNA in the control 
group. With this phase III TwiCs study we want to identify a group of patients at high risk for re-
currence after resection of the primary tumour based on detectable ctDNA and to show a benefi-
cial effect of adjuvant chemotherapy in this high risk group of patients on recurrence rate and 
overall survival. We expect to present a clinically relevant decrease in recurrence rate in ctDNA 
positive patients treated with adjuvant chemotherapy as compared to patients with detectable 
ctDNA who will only receive follow-up according to the current guideline. We expect that this will 
translate into improved overall survival. 
 
Preliminary results of our own research 
The departments of Medical Oncology, Surgery and Pathology of the Erasmus MC closely collabo-
rate and have pioneered and gained expertise in the pre-analytical conditions and optimised isola-
tion and molecular analyses of ctDNA. This resulted in multiple publications and the diagnostic 
implementation of NGS Oncomine cell-free DNA (cfDNA) assays (Thermo Fisher Scientific) for can-
cer patients since 2016.(28, 36-44) In a research setting, there is ample experience with breast 
cancer samples (Everolimus Biomarker study), colorectal cancer (CRC-IMPACT) and colorectal liver 
metastases samples (MIRACLE-study) that have been sequenced by the Department of Pathology 
using Oncomine assays. For example, the KWF funded MIRACLE-study aims to establish whether 
determination of cell-free DNA (cfDNA) in patients with isolated colorectal liver metastases under-
going hepatic resection can discriminate between patients showing a recurrence within 1 year 

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from those who do not. The presence of cfDNA in peripheral blood is analysed before and/or after 
hepatic resection to determine the association between ctDNA-based detection of MRD and clini-
cal outcome. Currently, the MIRACLE study has reached full accrual of 240 participants and first 
results were presented at ASCO 2024 (45).  
 
We briefly summarised the main findings of our completed projects: 
• 
We showed that next generation sequencing (NGS) and digital polymerase chain reaction 
(PCR) analyses are both tools to detect ctDNA by identification of tumour-specific mutations in 
circulating free DNA from blood of metastatic breast and colorectal cancer patients.(28, 36, 37, 
41) 
• 
We optimised pre-analytical variables such as the blood collection, demonstrating that ctDNA 
levels remain stable over time when blood is collected in BCT and CellSave preservative tubes.(39) 
Blood collected in standard EDTA tubes, however, will result in a decrease in the fraction of ctDNA 
over time as shown indicated by lower variant allele frequencies at 96-h samples compared to 1-h 
samples. 
• 
We pioneered different NGS approaches (38, 46, 47) to characterize mutations and measure 
accurately and sensitively ctDNA fractions. These include the standard customised Ampliseq pan-
els (Thermo Fisher Scientific) (38, 47) the OnTarget assay to enrich for mutant alleles by synchro-
nous coefficient of drag alteration technology (Boreal Genomics, Vancouver, Canada)(46), and re-
cently the Oncomine cfDNA assays with unique molecule identifiers (UMIs) (Thermo Fisher Scien-
tific).(28) The UMIs help to remove false positives due to library preparation and sequencing arte-
facts, resulting in more sensitive detection of mutations than standard NGS workflows. This ena-
bles us to detect ctDNA fractions as low as 0.1% in 20ng of circulating free DNA. 
• 
We did a comparison study on expression of cfDNA in terms of variant allele frequency ver-
sus number of mutant molecules.(44) 
• 
We demonstrated the feasibility of the Oncomine cfDNA colon assay in a cohort of meta-
static colorectal cancer patients receiving anti-EGFR (cetuximab) monotherapy.(28) We showed 
that mutations related to therapy resistance emerge in ctDNA during treatment with anti-EGFR 
monoclonal antibodies. In addition, as described above, in the MIRACLE cohort we used the On-
comine cfDNA colon assay to pre-operatively determine the mutations of interest for follow up 
analyses post-surgically. In this cohort we observed a clear difference in recurrence-free survival 
between MRD-positive and MRD-negative patients (figure 1). 
• 
Together with the Department of Pathology, Oncomine cfDNA assays and digital PCR anal-
yses are now used in a routine diagnostic setting in the Erasmus MC for lung cancer patients.  
 
 
Figure 1 – Recurrence-free survival 
after surgical removal of colorectal 
liver metastases in patients with and 
without post-operatively detectable 
ctDNA. 
 

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3.3 Mechanism of action, Drug class 
A summary of all products and its mechanisms of actions can be found in chapter 5 the SPC of 
capecitabine, oxaliplatin, leucovorin and 5-fluorouracil. 
3.4 Rationale for Dose Regimen/Dose Justification 
Description and justification of route of administration and dose is provided in chapter 8, which 
are all according to the standard of care as prescribed in the Dutch Guidelines. 
 
4. STRUCTURED RISK ANALYSIS  
4.1 Potential issues of concern 
4.1.1 Level of knowledge about mechanism of action 
The most concerning medication that is administered to patients allocated to the interven-
tion arm in this study is the chemotherapeutic agent oxaliplatin. Oxaliplatin is a third genera-
tion diaminocyclohexane (DACH) platinum compound that forms mainly intrastrand links be-
tween two adjacent guanine residues or a guanine and an adenine, disrupting DNA replica-
tion and transcription.(48) The details underlying the effects of oxaliplatin remain poorly un-
derstood. A summary of all products and its mechanisms of actions can be found in chapter 5 
the SPC of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil. 
 
4.1.2 Previous exposure of human beings 
Several clinical studies have investigated the safety CAPOX/FOLFOX, and thousands of pa-
tients have been treated with this combination treatment in the investigated popula-
tions.(17, 49) This regimen is consider safe and is registered in the Netherlands for patients 
with colorectal cancer in the adjuvant setting. A summary of all products can be found in the 
SPC of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil. 
 
4.1.3 Induction of the mechanism in animals and/or ex-vivo 
A summary of findings regarding the primary or secondary mechanisms of each interven-
tional product can be found in the SPC of capecitabine, oxaliplatin, leucovorin and 5-fluor-
ouracil.  
 
4.1.4 Selectivity of the mechanism  
A summary of findings regarding mechanism to target tissue of each interventional product 
can be found in the SPC of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil.  
 
4.1.5 Analysis of potential effect 
An analysis of the potential effect of each interventional product can be found in the SPC of 
capecitabine, oxaliplatin, leucovorin and 5-fluorouracil 
 
4.1.6 Pharmacokinetic considerations 
Pharmacokinetic parameters can be found in chapter 5 of the SPC of capecitabine, oxali-
platin, leucovorin and 5-fluorouracil. 
 

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4.1.7 Predictability of effect 
The clinical effect of CAPOX/FOLFOX is largely investigated, and the side-effects are well-
known (see 4.4 of the SPC of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil). Physi-
cians treating patients in this study are informed of these effects, and are considered to be 
predictable. 
 
4.1.8 Interaction with other products 
See paragraph 4.5 of the SPC of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil. 
 
4.1.9 Managing of effects 
Most side effects of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil are manageable. 
See 4.9 of the SPC of capecitabine, oxaliplatin, leucovorin and 5-fluorouracil for a detailed de-
scription of antidotes, antagonists and countermeasures. 
 
4.1.10  Study population 
The study population consists of patients with colorectal cancer that are eligible for 
chemotherapy, at the discretion of the treating physician. This means that all subjects will be 
screened for eligibility by a medical oncologist. The use of CAPOX/FOLFOX is well investigated 
in this population, and the side-effects are well-known. No pregnant women will be included 
in this study. 
 
4.2 Overall synthesis of the direct risks for the research subjects 
In the experimental group, patients will be treated with adjuvant CAPOX chemotherapy or with 
adjuvant FOLFOX chemotherapy. The combination chemotherapy schedule of CAPOX and FOLFOX 
are both registered products, and are commonly administered in the adjuvant setting in current 
practice for colorectal cancer. Therefore, the risks and toxicity of the used adjuvant chemotherapy 
are well-known. The mechanism of action is well-understood, its side-effects can be managed 
well, and the potential effect has been proven, for instance in palliative setting and in the adju-
vant setting of stage II and III colon cancer. Adjuvant chemotherapy for rectal cancer is standard 
of care in several countries, and although not recommended in the Dutch guidelines, population 
based studies from the Netherlands reveal that adjuvant chemotherapy is administered up to 29% 
of the patients with stage III rectal cancer.(50) All subjects in this trial will be adults. The extra po-
tential risks of adjuvant chemotherapy are acceptable in our opinion as we hypothesise that a sig-
nificant improvement in disease-free survival and overall survival can be obtained. Based on the 
guideline by the NFU (Dutch Federation of University Medical Centers) about quality insurance in 
human research (“Kwaliteitsborging van mensgebonden onderzoek”) we qualify the risk of this 
study as ‘slight risk (research involving a medical product)’ (moderate chance of slight damage). 
The most bothering toxicity of this regimen is sensory neuropathy, due to oxaliplatin. This neurop-
athy can be irreversible. Currently, we are not able to predict the chance of neuropathy upfront, 
and there are no effective drugs to prevent or treat this neuropathy. Trials to resolve this problem 
are ongoing. One year after treatment grade 2 neuropathy is 4.6% and grade 3 neuropathy is 
1.3%.(51) Severe and possible lethal toxicity of fluoropyrimidines is partially prevented by upfront 
testing for DPD-deficiency. Treatment-related mortality is <0,5%. The risk of the withdrawal of ex-
tra tubes of blood during regular blood withdrawal in all study participants is negligible. The obvi-
ous benefit for participants enrolled in this trial is the potential chance to reduce their risk of re-
currence with adjuvant chemotherapy. Subsequently, this will also increase their chance of 
achieving improved overall survival and quality of life. Thus, the investigators consider the extra 

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potential risk for patients in the experimental arm to be acceptable, hypothesising that a signifi-
cant improvement in outcomes can be obtained with additional chemotherapy in the specific pa-
tient population. 
 
5. OBJECTIVES AND ENDPOINTS 
 
Objective(s) 
Endpoint(s) 
Primary objective(s) 
Endpoint for the primary objective(s) 
• 
To investigate whether the disease-free 
survival in patients with rectal cancer 
who have detectable ctDNA after pri-
mary tumour resection, can be im-
proved by administration of adjuvant 
chemotherapy. 
• 
Primary endpoint of the study will be 
disease-free survival, calculated from 
the date of surgery to the date of 
progression (recurrence) or death from 
any cause of the patient, whichever 
occurs first. Patients who did not 
experienced a recurrence and who are 
still alive at the end of the study will be 
censored at the date of last contact. 
Secondary objective(s), if applicable 
Endpoint(s) for secondary objective(s), if 
applicable 
• 
To investigate whether overall survival 
of rectal cancer patients with detecta-
ble ctDNA after surgery can be im-
proved with adjuvant chemotherapy. 
• 
Overall survival will be calculated from 
the date of surgery to the date of death 
from any cause of the patient. Patients 
who are still alive at the end of the 
study will be censored at the date of 
last contact. 
• 
To compare all (eligible) patients in the 
control group to patients in the experi-
mental group who actually started the 
adjuvant chemotherapy by a per proto-
col analysis (for pure treatment effect) 
for disease-free and overall survival. 
• 
In addition to intention-to-treat analysis 
to estimate the effect size, also a per-
protocol analysis (for pure treatment 
effect) will be carried out for disease-
free survival and overall survival. This 
analysis will compare all (eligible) 
patients in the control group with those 
patients in the experimental group who 
actually started the adjuvant 
chemotherapy. 
• 
To compare the effect of adjuvant 
chemotherapy on quality of life. 
• 
Quality of life will be assessed in both 
groups by obtaining questionnaires 
already provided by the PLCRC cohort 
study. This to compare the effect of 
adjuvant chemotherapy on quality of 
life. In the whole study period of 4 
years there are 8 quality of life 
measurements. Quality of life is 
assessed with the following 
questionnaires: EQ-5D-5L, QLQ-C30 and 
QLQ-CR29. 

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• 
To compare the disease-free survival 
and overall survival of patients with de-
tectable ctDNA but without receiving 
adjuvant chemotherapy with patients 
with undetectable ctDNA. 
• 
The disease-free survival and overall 
survival of ctDNA negative patients will 
also be collected and analysed to 
confirm the robustness of post-
operative ctDNA as biomarker in rectal 
cancer. 
• 
To compare the clearance of ctDNA of 
the patients who received adjuvant 
chemotherapy in the experimental 
group with the patients in the control 
group. 
• 
The results of the ctDNA analysis of the 
blood samples taken at the first follow-
up of the randomised patients who 
were ctDNA positive after surgery will 
be compared between the patients who 
received adjuvant chemotherapy and 
those who did not. 
• 
To investigate the co-occurrence of 
ctDNA in peripheral blood at the timing 
of detection of recurrent disease on im-
aging. 
• 
Within the group of patients with de-
tectable ctDNA after surgery, the pro-
portion of patients with detectable 
ctDNA in the peripheral blood at the 
timing of detection of recurrent disease 
on imaging will be assessed. 
Exploratory objective(s), if applicable 
Endpoint(s) for exploratory objective(s), if ap-
plicable 
• 
To determine the mutational profile 
and ctDNA levels in ctDNA-positive pa-
tients.  
• 
The proportion of ctDNA-positive pa-
tients with specific mutational profiles. 
• 
Quantitative ctDNA levels 
• 
To identify and analyse specific muta-
tions detected in ctDNA and their corre-
lation with clinical and pathological pa-
rameters, as well as time to recurrence. 
• 
Correlations between ctDNA mutational 
profiles and clinical/pathological param-
eters and time to recurrence. 
• 
To compare the mutational status of 
ctDNA with the mutational profile of 
available tumour tissue in patients, as-
sessing concordance and potential dis-
crepancies. 
• 
Concordance rate (%) between muta-
tions identified in ctDNA and matched 
tumour tissue. 
 
 
 

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6. STUDY PLAN AND DESIGN 
6.1  Trial Design 
Trial within cohorts (TwiCs) design 
This study is designed as a randomised controlled trial within a prospective cohort of patients. The 
study design and randomisation progress is demonstrated in Figure 2.  
 
 
 
 
Figure 2. Flowchart of the REACT study in the trial within cohorts (TwiCs) design.  
 

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6.2  Number of Patients 
With a detection rate of 15% for ctDNA after surgery in high-risk rectal cancer, 1373 patients’ blood 
samples need to be analysed for ctDNA to include all patients with detectable ctDNA (n=206), 103 
patients with detectable ctDNA for each group.  
 
6.3 Overall study duration and follow-up 
The anticipated overall study duration for each subject is 3 months (12 weeks), corresponding to the 
period of investigational treatment with adjuvant chemotherapy. Adjuvant chemotherapy consists of 
6 cycles of 5FU/folinic acid and oxaliplatin (FOLFOX) every 2 weeks, or 4 cycles of capecitabine and 
oxaliplatin (CAPOX). Following study participation, postoperative follow-up will be conducted in 
accordance with the Dutch Colorectal Cancer Guidelines, which currently recommend monitoring for 
up to 5 years after rectal surgery. 
 
6.4 Patient participation 
The patient federation ‘Stichting Darmkanker” was asked for assistance with the development of our 
study design and the informed consent form. Constructive meetings were held to ensure that all 
written information used in this study is understandable for patients with different educational lev-
els. The patient federation supports our study design and will remain involved during further steps of 
this application. We have included a patient who had surgery and chemotherapy for rectal cancer in 
the past, to act as subject who has expertise by experience. Together with the other involved collab-
orators of Stichting Darmkanker, the principal investigators and the coordinating investigator, we 
have created an advisory group that will be involved from the beginning to end of the REACT study. 
During the trial, regular meetings and focus groups will be held with the advisory group and feedback 
will be provided. Where necessary, adjustments will be made. Also, the contact information from this 
advisory group will be included in the patient information letter (PIF) and the REACT protocol, so that 
this group can both support the participants of this trial, as well as provide feedback for the REACT 
study team.  
 
 
 

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7. STUDY POPULATION 
7.1 Population  
Patients included in PLCRC (MEC-2016-369), with high-risk rectal cancer who underwent surgery, 
fit to receive adjuvant chemotherapy (WHO performance score 0-1) will be selected for inclusion 
in this study. High-risk rectal cancer is defined as either; treated with neoadjuvant therapy; and/or 
clinical T4; and/or clinical N+. The aim is to include all patients within three years. 
 
7.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following criteria: 
• 
Detectable ctDNA in the postoperative blood sample 
• 
Age ≥ 18 years 
• 
WHO performance score 0-1 
• 
Informed consent for PLCRC with specific consent for additional blood withdrawals and 
offering of future experimental research  
• 
Informed consent for the REACT trial. 
• 
Histological confirmed rectal cancer; either treated with neoadjuvant (chemo)radiotherapy, 
and/or clinical/pathological T3/T4 and/or N+ in case no neoadjuvant therapy was 
administered.  
• 
Eligible to receive treatment with combination adjuvant chemotherapy (CAPOX/FOLFOX) 
according to the treating physician. 
• 
Mentally competent and able to read and understand Dutch language. 
 
7.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation in 
this study: 
• 
Metastatic disease 
• 
Another malignancy in previous 5 years, with the exception of treated carcinoma in situ or 
skin cancer other than melanoma 
• 
Incomplete primary tumour resection (R1 or R2 resection) 
• 
Contra-indication for fluoropyrimidines or oxaliplatin 
• 
Neoadjuvant oxaliplatin based systemic treatment, e.g. treated with the RAPIDO regimen 
consisting of short course radiotherapy followed by 6 cycles of CAPOX or 9 cycles of FOLFOX 
prior to surgery 
• 
Patients with a clinical complete response, who will not undergo surgery. 
• 
Pregnant and lactating women 
• 
History of psychiatric disability judged by the investigator to be clinically significant, 
precluding informed consent or interfering with compliance of the intervention group 
• 
Serious concomitant systemic disorders that would compromise the safety of the patient or 
his/her ability to complete the study, at the discretion of the investigator 
• 
Serious infections (uncontrolled or requiring treatment) 
• 
Current or recent (within 28 days prior to randomisation) treatment with another 
investigational drug or participation in another study interfering with the primary endpoint. 
 
7.4 Vulnerable populations and clinical trials in emergency situations 
Not applicable 

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8. STUDY TREATMENTS 
Treatment of patients in the experimental group: 
 
Adjuvant chemotherapy is not a part of standard care in European guidelines, however in  several 
countries it is the standard of care. The aim of this study is to use standard regimens of combined ad-
juvant chemotherapy, known from current treatment of colon cancer, only in high risk patients based 
on detectable ctDNA after surgery.  
Adjuvant chemotherapy is initiated preferably within 8 weeks after surgery and no longer than 12 
weeks after surgery. Adjuvant chemotherapy consists of 4 cycles CAPOX every 3 weeks or 6 cycles 
FOLFOX every 2 weeks.  
 
The regimens and dosing schedules of CAPOX and FOLFOX are : 
• 
CAPOX (4 cycles every 3 weeks) 
- 
Capecitabine: 1000 mg/m2 bid orally day 1-14  
- 
Oxaliplatin 130 mg/m2 iv. day 1 in 2 hours 
• 
FOLFOX (6 cycles every 2 weeks) 
- 
5FU: 400 mg/m2 iv. day 1, followed by continuous infusion 2400 mg/m2 for 46 h 
- 
Folinic acid: 400 mg/m2 day iv. day 1 
- 
Oxaliplatin: 85 mg/m2 iv. day 1 
 
Note: Recent studies have demonstrated non-inferiority for three months versus six months of adju-
vant chemotherapy in patients with colon cancer. (52-54) The adverse effects of oxaliplatin are well 
known in current practice. Currently this regiment is incorporated in the Dutch national guideline, 
therefore we choose for 3 months CAPOX or FOLFOX regimen of adjuvant chemotherapy. 
 
8.1 Investigational Medicinal Product(s) (IMP(s)) 
8.1.1 Name and description of the IMP 
In the present study, patients in the experimental group will receive adjuvant chemother-
apy with CAPOX or FOLFOX. These are established chemotherapeutic regimens that are 
well characterized and known to be well tolerated by patients. 
• 
Capecitabine (chemical name: L01BC06) 
• 
Oxaliplatin (chemical name: L01XA03) 
• 
5-fluorouracil (chemical name: L01BC02) 
• 
Folinic acid (chemical name: V03AF03) 
 
All investigational products have a marketing authorisation and are used in the authorised 
form for the authorised indication. 
 
8.1.2 Status of development of the IMP 
A summary of findings can be found in paragraph 4.1 of the SPC of capecitabine, oxaliplatin, 
folinic acid and 5-fluorouracil. 
 
8.1.3 Description and justification of dosage and route of administration  
Description and justification of route of administration and dose is provided above, which 
are all according to the standard of care as prescribed in the Dutch Guidelines. 
8.2 Comparator IMP(s) 
Not applicable 

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8.3 Placebo 
Not applicable. 
8.4 Auxiliary Medicinal Product(s) (AxMP(s)) 
Not applicable. No auxiliary medicinal products will be used in this study other than the 
investigational products as described above. 
 
8.4.1 Name and description of the AxMP 
Not applicable. 
 
8.4.2 Statement on authorisation and justification unauthorised AxMP (if applicable) 
Not applicable. 
 
8.4.3 Description and justification of dosage and route of administration  
Not applicable. 
 
8.5 Additional considerations for trials involving a medical device 
Not applicable 
 
8.6 Additional considerations for trials involving an in-vitro diagnostic or companion 
diagnostic 
The Oncomine™ Colon cfDNA Assay is performed in a laboratory for Molecular Diagnostics in the 
department of Pathology that is accredited according to ISO15189:2012 criteria of the Dutch 
Accreditation Council (RvA) and European IVD regulations (i.e. comply with the applicable General 
Safety and Performance Requirements as described in Annex I of the European Regulation EU 
2017/746). 
 
8.7 Preparation and labelling of the study treatment(s) 
All investigational products have a marketing authorisation and are used in the authorised form 
for the authorised indication. Preparation and labelling will be according to local standard. No 
specific labelling is performed in this study. 
 
 
 
 

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9. OTHER TREATMENTS AND RESTRICTIONS 
9.1 Concomitant therapy 
 
9.1.1 Permitted medication(s) 
During chemotherapeutic treatment patients will receive co-medication according to 
standard procedures of local site. 
 
9.1.2 Prohibited medication(s) 
At study initiation, patients should report their concomitant medications to their treating 
physician, if there are medications with interaction with CAPOX or FOLFOX the treating 
physician, if possible, can change or replace the concomitant medications of the patient. 
According to routine clinical practice, use of co-medication and escape medication to 
diminish side effects is allowed. Patients are recommended to contact their treating 
physician when adverse reactions occur according to routine clinical practice. 
 
9.2 Lifestyle restrictions 
9.2.1 Contraception measures 
Women who are pregnant or breastfeeding are excluded from participation in this study. Ad-
ditionally, women of childbearing potential are required to use highly effective contraception 
throughout the study period to prevent pregnancy. Male participants with female partners of 
childbearing potential must take measures to ensure that their partners do not become preg-
nant during the study. The investigator will provide detailed instructions regarding appropri-
ate contraceptive methods, which should be discussed with the participant’s partner. 
 
Management of Pregnancy During the Study 
If a female participant becomes pregnant during the study, she must immediately notify the 
investigator and discontinue participation. If the female partner of a male participant be-
comes pregnant during the study, the participant is encouraged to seek her consent to in-
form the investigator. Upon receiving consent, the investigator will arrange for additional 
monitoring of the pregnancy and may collect relevant information regarding the progress 
and outcome of the pregnancy from healthcare providers. Such monitoring and data collec-
tion will only occur if explicit consent is provided by the pregnant partner. 
 
9.2.2 Other requirements 
Not applicable 
 
10. TRACEABLILITY, STORAGE, ACCOUNTABLILITY AND COMPLIANCE 
 
10.1 Traceability and storage of the study treatment(s)? 
Not applicable, since all investigational products are used from commercial stock in the authorised 
form for the authorised indication. 
 
10.2 Accountability of the study treatment(s) and compliance 
Not applicable. 
 

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11. STUDY ASSESSMENTS AND PROCEDURES 
11.1 Screening procedure 
Participating PLCRC centers can register patients for screening for the REACT study in the online 
Ldot software that is used by the PLCRC. If the patient is eligible for REACT and has given written 
informed consent for PLCRC, including informed consent for additional blood withdrawals and of-
fering of future studies, the patient can be added to the REACT sub-study in Ldot. The REACT 
study team will then automatically be informed that a new patient has been added to the screen-
ings list. At the same time, instructions about the timing and collection of the post-operative 
blood samples will be send out to the treatment team of the patient. Within 14-28 days after rec-
tal surgery, blood collected in CellSave tubes will be send to the laboratory of the Medical Oncol-
ogy department of Erasmus MC for ctDNA analysis. 
11.2 Randomisation, blinding and treatment allocation 
If patients have detectable ctDNA after surgery and are eligible for the REACT study, patients will 
be randomised 1:1 to the control group or intervention group within the database. Randomisation 
will be performed by the REACT research team using the LDOT software. The REACT research 
team from Erasmus MC Cancer Institute will receive the results from the randomisation and will 
inform the treating physician of the patient when included in the experimental group.   
 
Control group: standard of care group 
In this group, patients and their treating physician will not be informed about this study. Patients 
will not be informed about the randomisation to the control group, according to the TwiCs 
principle, for which patients already gave informed consent within the PLCRC study cohort. 
Patients and their treating physician will not be informed about the allocation in the control group 
and the results of the ctDNA analysis. Patients will not be offered adjuvant chemotherapy (= 
standard of care). All patients in the control group will receive standard of care and standard 
follow up, there are no consequences for this group. 
 
Intervention group: adjuvant chemotherapy 
In this group, patients will receive information about this study and randomisation to the 
intervention group by their treating physician. Patients will be informed about previous ctDNA 
analyses within the PLCRC study and the meaning of ctDNA. The finding of detectable ctDNA in 
their peripheral blood samples and the possible high risk on recurrence with detectable ctDNA 
will be discussed. Patients will be informed about the possibility to receive adjuvant 
chemotherapy and the potential beneficial effect of adjuvant chemotherapy will be explained. 
Patients who are interested in receiving adjuvant chemotherapy, will be referred to the medical 
oncology department. After extensive counselling by a medical oncologist, the patient will decide 
whether they want to receive adjuvant chemotherapy or not. If patients want to receive adjuvant 
chemotherapy, informed consent will be obtained and adjuvant chemotherapy (CAPOX or 
FOLFOX) will be administered.. After completing the adjuvant chemotherapy, patients will receive 
standard-of care follow-up.. If patients refuse adjuvant chemotherapy, they will receive standard 
follow-up by their own treating physician. 
 
11.3 Study procedures and assessments 
Prospective Dutch ColoRectal Cancer cohort 
All patients with colorectal cancer can be included in the PLCRC cohort study (MEC-2016-369) in 
which clinical data, blood, tissue and Patient Reported Outcomes (PROMs) are registered from 

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diagnosis until death of stage I-IV colorectal cancer patients.(55) PLCRC was initiated as a multi-
centre study in 2015. The aim of PLCRC is to improve outcomes of colorectal cancer patients by 
providing a research infrastructure. 
 
Within PLCRC several observational and interventional sub-studies are currently being conducted, 
either according to the TwiCs design or not. From patients participating in the PLCRC cohort that 
meet the inclusion criteria, and fit for adjuvant chemotherapy (WHO performance score 0-1), who 
gave consent for additional blood withdrawal within the PLCRC study, blood samples will be col-
lected within 14 – 28 days after surgery. These blood samples will be analysed for detectable 
ctDNA. Patients with undetectable ctDNA will not be randomised.  In case of detectable ctDNA 
patients will be randomised in a 1:1 ratio to a control group (standard of care group consisting of 
follow-up) and an experimental group who will be offered adjuvant chemotherapy. In case of un-
detectable ctDNA or detectable ctDNA and allocation to the control group, test reports will not be 
reported to the patient and treating physician (see “Ethical considerations” (chapter 11)). 
 
Patients that will undergo resection of rectal cancer with curative intent and meeting the inclu-
sion criteria will undergo blood withdrawal for ctDNA analysis 2-4 weeks after surgery. The ctDNA 
analysis will take approximately 3 weeks. If there is detectable ctDNA after surgery and patients 
are eligible according to in- and exclusion criteria, these patients will be randomised according to 
the TwiCs design. For those patients with detectable ctDNA who are randomized to the control 
group, no additional informed consent is required; patients already gave informed consent to act 
as a control group with the PLCRC informed consent. They will receive standard of care and nor-
mal follow up. Patients with detectable ctDNA and randomised to the experimental group will be 
asked for written informed consent at the participating hospital after counselling about this study 
and the randomisation for the intervention. They are informed about the detected ctDNA and the 
associated high risk of recurrence. They are offered adjuvant chemotherapy with the aim to re-
duce the chance of disease recurrence. In this case, they will be counselled about adjuvant chem-
otherapy by a medical oncologist. Patients will decide whether to receive adjuvant chemotherapy 
or not. If they want to undergo adjuvant chemotherapy, this will be initiated as soon as possible, 
at a maximum of 12 weeks from surgery. 
 
CtDNA analyses 
Blood samples will be withdrawn at times of regular blood withdrawal. Patients have given in-
formed consent in PLCRC for these additional blood withdrawals for research purposes with a 
maximum of 16 tubes per year (no more than 4 tubes at a time). 
Timing of withdrawal of blood samples for baseline ctDNA testing are: 
- Within 14-28 days after surgery (2 tubes of 10 mL) 
 
Blood collected in CellSave tubes from patients of all PLCRC participating hospitals will be send to 
the Erasmus MC - Cancer Institute. The Medical Oncology liquid biopsy laboratory will process the 
blood within 96h after blood drawing to obtain plasma according to our standard guidelines.(39) 
It is expected that blood of about 5 patients will arrive on a weekly basis.  
 
At week 1 work-day 5, circulating free DNA (cfDNA) will be isolated from 4 ml of plasma for all 
samples received in that week using the qiaAMP circulating nucleic acids kit (Qiagen) following 
the manufacturer’s instructions. In the second week, the obtained cfDNA will be quantified and at 

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least 10 ng will be used for the library preparation of the Oncomine cfDNA colon panel (Ther-
moFisher). This assay contains primers to sequence 49 amplicons for hotspot regions of 14 colon 
cancer relevant genes (AKT1, APC, BRAF, CTNNB1, EGFR, ERBB2, FBX7, GNAS, KRAS, MAP2K1, 
NRAS, PIK3CA, SMAD4, and TP53) to identify 236 hotspot mutations and indels. After quality con-
trol of the library preparations, these preps will be sequenced on a Ion Torrent S5 sequencer at 
high coverage (more than 20.000 reads depth).  
 
At week 2 work-day 5, base calling, read mapping and variant calling are performed using the Tor-
rent Suite (BaseCaller), tmap and torrent variant caller, (Thermofisher), with default settings for 
cfDNA.  
 
In week 3, the reports will be evaluated and authorized by a team of ctDNA experts. We will retain 
variants which have a variant allele frequency (VAF) above the limit of detection based on the mo-
lecular coverage of the specific variant position (provided by torrent variant caller software).Most 
hotspot oncogenic mutations evaluated by our targeted panel are not suspect to clonal haemato-
poiesis of indeterminate potential (CHIP; i.e. the presence of a clonally expanded hematopoietic 
stem cell caused by a leukemogenic mutation in individuals without evidence malignancy). How-
ever when CHIP is suspected based on the list of canonical CHIP genes (PMID: 31768066) together 
with the observed VAF(), additional germline DNA analyses will be performed to determine 
whether the detected mutation originates from germline- or tumor DNA.    
 
In case a blood sample contains ctDNA, identified by tumour-specific mutations, the laboratory 
staff will contact the Erasmus MC research team before the end of week 3, and they will handle 
the further actions of randomisation and, when allocated to experimental arm, inform the treat-
ing physician of the ctDNA detected patients 
 
The additional blood samples of the ctDNA positive patients that will be collected at first follow-
up after surgery and after recurrent disease will immediately be send to the Medical Oncology la-
boratory of Erasmus MC as well. Here they will be processed to plasma within 96 hours after 
blood withdrawal and then stored in the REACT biobank. After completion of the intervention 
part of this trial we will evaluate the presence and level of ctDNA at the time of the first return 
visit and at time of recurrence. For ctDNA analysis, the same steps will be followed as described 
above. If a targeted PCR is deemed feasible based on the baseline ctDNA analysis results, this will 
be performed instead of the Oncomine colon assay to reduce costs. We already have experience 
in this based on the MIRACLE study, which has provided us with several prepared assays for tar-
geted PCR analysis.    
 
Quality of life 
Quality of life will be assessed in both groups by obtaining questionnaires already provided by the 
PLCRC cohort study. This to compare the effect of adjuvant chemotherapy on quality of life. 
Patients participating in PLCRC optionally receive validated quality of life questionnaires. These 
questionnaires are available for this study as well. We will assess and compare quality of life in the 
intervention group compared to the control group. Questionnaires are obtained within PLCRC 
(digital) at baseline, 3 months, 6 months, 1 year, 1.5 years, 2 years and yearly after. In the whole 
study period of 4 years there are 8 quality of life measurements. We expect a response rate of 
80%, based on current results from the PLCRC study board. Quality of life is assessed with the 
following questionnaires: EQ-5D-5L, QLQ-C30 and QLQ-CR29. 

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Follow-up 
Follow up starts from the day of surgery in both groups. Follow up for patients in the control and 
experimental group will be investigations according to Dutch national and participating hospital 
guidelines. Thus, follow-up for evaluation of recurrences will be within the same time intervals for 
both the control group and intervention group, despite adjuvant chemotherapy treatment in the 
intervention group. All follow-up is according to the Dutch national and hospital guidelines with 
blood sampling of tumour marker CEA and imaging by ultrasound or CT-thorax/abdomen. 
 
Additional blood samples 
At the first planned follow-up visit after rectal surgery according to the guidelines (approximately 
after 3-4 months), two additional CellSave tubes (10 mL) will be collected from all baseline ctDNA 
positive patients. In case a patient has received adjuvant chemotherapy, the blood withdrawal 
must take place at least 14 days after completion of chemotherapy. Furthermore, in case of recur-
rent disease another two CellSave tubes (10 mL) will be collected during the next scheduled blood 
withdrawal of all patients who had detectable ctDNA after surgery.  
 
Importantly, in the PLCRC study patients can withdraw their informed consent for additional 
blood samples at any time. Therefore, informed consent will be checked before blood withdrawal 
and patients who no longer have informed consent will be excluded from these additional blood 
samples. 
 
Data collection for PLCRC and REACT  
The clinical data for PLCRC and (sub)studies are collected by data managers of the Netherlands 
Comprehensive Cancer Organization (IKNL). The data are registered in the Netherlands Cancer 
Registry (NCR) according to the standard protocols and definitions of IKNL. The observational data 
will prospectively be collected from medical charts and pathology reports.  
 
All patients that underwent blood withdrawal for ctDNA analysis for the REACT study, will be reg-
istered in a study specific secured database with their unique pseudonymized REACT study num-
ber (REACT-SITE-XXXX) and PLCRC study number (PICNICXXXXXXXX). Data about timing and 
amount of blood withdrawals for ctDNA analysis will be recorded, as well as the results of the 
ctDNA analysis. From the patients in the intervention group, additional information about the tim-
ing and dosage of the adjuvant chemotherapy will be registered. No patient identifying data such 
as date of birth or date of surgery will be stored in the database. The REACT study team will man-
age the data management of the REACT database. 
 
Halfway and at the end of the study, the clinical data of all patients that underwent blood with-
drawal for ctDNA analysis for the REACT study will be extracted from the NCR after submitting a 
data request at IKNL. The clinical data will include all patient characteristics, tumour characteris-
tics and oncological outcomes. The received dataset will be matched to the study specific dataset 
based on the unique PICNIC numbers and then used for the final analysis.  
 
Survival data 
The survival data of the Netherlands Cancer Registry will be used to ascertain the exact date that 
a certain patient has deceased and will be managed by the PLCRC. The Netherlands Cancer Regis-
try routinely collects cancer survival data through linkage with the “Municipal Personal Records 

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Database” (Dutch: Basisregistratie personen), which is organized in a specific law (Wet Basisregis-
tratie Personen, WBP). 
 
 
Data extraction from the study database 
Requested data can be extracted from the study databases of PLCRC and REACT by exporting data 
to Excel, SPSS and/or SAS. This extraction will contain data from all patients or, if requested, from 
a selection of patients. Patients participating in the REACT study are given an unique sub study 
patient number (REACT – SITE – study number). Data will only be exported coded (i.e. without any 
identifying information such as patient name, initials). 
 
11.3.1 Efficacy assessments 
The efficacy of the studied intervention of administration of adjuvant chemotherapy in 
ctDNA positive patients after rectal surgery is determined by the primary and secondary 
outcome measures of disease-free-survival and overall survival, respectively. In addition, the 
clearance of ctDNA of the patients who received adjuvant chemotherapy in the experimental 
group will be compared with the patients in the control group. The methods and timing of 
the analysis of these parameters can be found in chapter 14.   
 
11.3.2 Safety assessments 
Adverse events of established chemotherapeutic regimens (CAPOX, FOLFOX) after surgery in 
patients with colorectal cancer is well investigated. Therefore, no study specific safety 
assessments will be performed during the trial besides the standard of care procedures 
according to the Dutch Guidelines. The reporting of serious adverse events is further 
discussed in chapter 13. 
 
 
 
 
 
 
 
 
 
 

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12. STUDY DISCONTINUATION AND COMPLETION 
 
12.1 Definition End of Trial 
The end of the study is defined as the last included patient’s last visit. 
 
12.2 Criteria for temporary halt and early termination of the clinical trial 
The Sponsor may decide to terminate the study prematurely based on the following criteria: 
• 
There is evidence of an unacceptable risk for study patients in accordance with the princi-
ple investigator and/or study team (i.e. safety issue); 
• 
There is reason to conclude that it will not be possible to collect the data necessary to 
reach the study objectives and it is therefore not ethical to continue enrolment of more 
patients; for example insufficient enrolment that cannot be improved. 
The Sponsor will promptly notify all concerned investigators, the Ethics Committee(s) and the reg-
ulatory authorities of the decision to terminate the study. The Sponsor will provide information 
regarding the time lines of study termination and instructions regarding treatment and data col-
lection of enrolled patients.  
 
12.3 Discontinuation/withdrawal of individual subjects 
Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any conse-
quences. The investigator can decide to withdraw a subject from the study for urgent medical rea-
sons. 
 
Specific criteria for withdrawal  
Study treatment (adjuvant chemotherapy) will be terminated in case of severe adverse reactions, 
not manageable with standard interventions. These patients will be analysed according to the in-
tention-to-treat principle. Patients in in the experimental group that did not started with adjuvant 
chemotherapy, will not be included in the per-protocol analysis.  
 
Replacement of individual subjects after withdrawal 
Subjects that are withdrawn from the study will not be replaced. 
 
Follow-up of subjects withdrawn from treatment 
Clinical data from patients that are withdrawn from treatment will be followed within the PLCRC 
cohort study. Data from patients that are lost to follow-up will be censored to the last date of 
data collection by data-managers. Subjects redrawn from treatment will receive standard-of-care 
treatment.   
 
12.4 Arrangements for subjects after their participation in the clinical trial ended 
Not applicable. Patients continue standard care according to Dutch guidelines. 
 

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13. SAFETY REPORTING 
13.1 Definitions 
13.1.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the 
study, whether or not considered related to the experimental intervention (CAPOX or FOL-
FOX).  
 
13.1.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes 
listed above due to medical or surgical intervention but could have been based 
upon appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
13.1.3 Suspected unexpected serious adverse reactions (SUSARs) 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. The event must be serious; 
2. There must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of the 
administered dose; 
3. The adverse reaction must be unexpected, that is to say, the nature and severity of the 
adverse reaction are not in agreement with the product information as recorded in the 
Summary of Product Characteristics (SPC).  
 
13.2 Recording of AEs/SAEs/SUSARS 
Chemotherapy is a standard of care in colorectal cancer patients, and AEs and SAEs are well-
known. Adverse events of established chemotherapeutic regimens (CAPOX, FOLFOX) after surgery 
in patients with colorectal cancer is well investigated and will not be recorded. SAEs of patients 
allocated to the experimental group do not have to be reported immediately (<24 hours), but 
have to be reported in the study file and will be reported in the annual progress report. Only SAEs 
within 1 year after start of adjuvant chemotherapy have to be reported. Information on type of 
SAE and grade according the Common Terminology Criteria for Adverse Events (CTCAE) Version 
5.0 will be reported.(48) 
 
The sponsor will report the SAEs to the European Medicines Agency (EMA) via the Eudravigilance 
database, within 7 days of first knowledge for SAEs that result in death or are life threatening fol-
lowed by a period of maximum of 8 days to complete the initial preliminary report. All other SAEs 
will be reported within a period of maximum 15 days after the sponsor has first knowledge of the 
serious adverse events. 
 

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All SUSARs should be reported in the electronic patient files as soon as possible. The local investi-
gator should report all SUSARs within 24 hours to the electronic patient files using a SUSAR form. 
The REACT study team will evaluate whether the SAE indeed qualifies as a SUSAR and will report 
these in the (e)CRF and sub-study database. All SUSARs of patients allocated to the experimental 
group will be reported in the annual progress report. SUSARs of patients allocated to the experi-
mental group do not have to be reported immediately because the toxicity and adverse effects of 
CAPOX and FOLFOX are well known and safety is not investigated in this study. 
13.3 Reporting of AEs and SAEs 
13.3.1 Reporting of SAEs by the investigator to the sponsor 
SAEs of patients allocated to the experimental group do not have to be reported immediately 
(<24 hours) by the investigator to the sponsor, but have to be reported in the study file and 
will be reported in the annual progress report. 
 
13.3.2 List of SAEs which do not require immediate reporting and procedure for reporting 
SAEs of patients allocated to the experimental group do not have to be reported immedi-
ately (<24 hours) by the investigator to the sponsor, but have to be reported in the study file 
and will be reported in the annual progress report. 
 
13.4 Follow-up of adverse events 
SAEs of patients allocated to the experimental group need to be reported till the end of study within 
the Netherlands, as defined in the protocol. They will be recorded in the CRF. There is no active fol-
low-up of SAEs by the central research team. AEs will not be reported. The treatment and follow-up 
of (S)AE will be according to the standard of care. 
 
13.5 Reporting of SUSARs by the sponsor to EudraVigilance  
The sponsor will report electronically and without delay to EudraVigilance all relevant information 
about any SUSAR (CTR: Article 42). 
 
The period for the reporting of SUSARs by the sponsor to EudraVigilance will take account of the 
seriousness of the reaction and will be as follows:  
− In the case of fatal or life-threatening SUSARs, as soon as possible and in any event not later than 
7 days after the sponsor became aware of the reaction (CTR: Article 42(2(a))); 
− In the case of non-fatal or non-life-threatening SUSARs, not later than 15 days after the sponsor 
became aware of the reaction (CTR: Article 42(2(b))); 
− In the case of a SUSARs which was initially considered to be non-fatal or nonlife threatening but 
which turns out to be fatal or life-threatening, as soon as possible and in any event not later than 
7 days after the sponsor became aware of the reaction being fatal or life-threatening (CTR: Article 
42(2(c))). 
 
Where necessary to ensure timely reporting, the sponsor may, in accordance with section 2.4 of 
Annex III, submit an initial incomplete report followed up by a complete report (CTR: Article 42(2)). 
13.6 Annual safety report 
Regarding investigational medicinal products other than placebo, the sponsor shall submit annually 
through CTIS to all Member States concerned a report on the safety of each investigational medicinal 
product used in a clinical trial (CTR: Article 43). 

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13.7 Unblinding procedures for safety reporting 
Not applicable 
 
13.8 Temporary halt for reasons of subject safety 
The sponsor will suspend the study if there is sufficient ground that continuation of the study will 
jeopardise subject health or safety. The sponsor will submit the notification through CTIS without 
undue delay of a temporary halt but not later than in 15 days of the date of the temporary halt. It 
shall include the reasons for such action and specify follow-up measures. The study will be 
suspended pending a further positive decision by the concerned member state (CTR: Article 38). The 
investigator will take care that all subjects are kept informed. 
 
13.9 Urgent safety measures and other relevant safety reporting 
Where an unexpected event is likely to seriously affect the benefit-risk balance, the sponsor and the 
investigator will take appropriate urgent safety measures to protect the subjects. In addition, the 
sponsor will notify the Member States concerned, through CTIS, of the event and the measures 
taken. That notification will be made without undue delay but no later than 7 days from the date the 
measures have been taken (CTR: Article 54). 
 
13.10 Data Safety Monitoring Board (DSMB)/Data Monitoring Committee (DMC) 
A Data and Safety Monitoring Board will not be installed. In the present study, patients in the experi-
mental group will be treated with established chemotherapeutic regimens (CAPOX, FOLFOX) that are 
well characterized and known for not harming patients. All subjects in this study will be adults. Based 
on previous data, it is not expected that the primary endpoint (disease-free survival) will be estab-
lished with statistical significance prior to the date of last enrolment. Premature termination of the 
study for reasons of superiority (or futility) is therefore unexpected. 
 
 
 
 
 
 
 

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14. STATISTICAL ANALYSIS 
 
14.1 Description of statistical methods 
All main analyses will be according to the intention to treat principle, i.e. patients will be analysed 
according to the study group they were assigned to; this also holds for patients randomised to the 
experimental group but who refuse to receive adjuvant chemotherapy. However, patients initially 
randomised but considered ineligible afterwards based on information that should have been availa-
ble before randomisation, will be excluded from all analyses. The main analysis addressing the pri-
mary endpoint will be done after 118 events, and is planned two years after the last included patient. 
Before this analysis, a statistical analysis plan will be prepared by the statistician  in consultation with 
the principal investigator. Other analyses of the secondary endpoints and/or involving safety anal-
yses could be performed when the needs arise and in discussion with the study coordinators. How-
ever, all other analyses except the primary analysis should be regarded as exploratory, and therefore 
only as hypothesis-generating. Analyses will be performed in R version 4.2.1 (https://www.r-pro-
ject.org/). 
 
14.2 Analysis sets 
All patients who underwent blood withdrawal for ctDNA analysis for the REACT study who meet the 
in- and exclusion criteria, will be included in the statistical analyses.  
 
14.3 Participant demographics and other baseline characteristics 
Patient characteristics: age, sex, length, weight, cTNM, type of surgery, type of neoadjuvant treat-
ment, pTNM, WHO performance status, type and duration of chemotherapy, dosing and dose adjust-
ments of adjuvant chemotherapy, serious adverse events, extramural vascular invasion, mesorectal 
fascia involvement, lateral lymph node involvement. 
 
14.4 Randomisation and blinding 
Randomisation will be performed within the online Ldot software platform. Following eligibility verifi-
cation by the REACT studyteam, patients with detectable ctDNA after rectal surgery will be random-
ized in a 1:1 ratio to either the control group or the experimental group. No stratification factors will 
be applied. Random block sizes of 4,6, or 8 will be used. 
 
14.5 Sample size, trail power and level of significance used 
In the Netherlands, rectal cancer is diagnosed in 3500 patients each year. The estimated proportion 
of patients with stage II or III rectal cancer is 65%. However, due to the nationwide bowel screening 
program which was implemented in the Netherlands in 2014, stage migration has been observed 
with detection of more early cancers, resulting in an estimated stage distribution of 50% for stage II-
III during the inclusion period. 
 
Of the patients with stage II-III disease, about 15% will have clinical complete response after neo-
adjuvant treatment and will not have surgery, therefore they cannot be screened. All other patients 
with meeting the inclusion criteria and detectable ctDNA post-surgery will be eligible for inclusion. 
The PLCRC study cohort will be used for inclusion of patients for the present study. Patients eligible 
for this study with detectable ctDNA will be randomised according to the TwiCs principle. For the 
control group no additional informed consent is required, only the experimental group needs 

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additional informed consent. With the use of the PLCRC cohort and its participating hospitals we 
expect to assure timely inclusion of 206 ctDNA positive patients, which are needed to assess the 
primary objective of this study.  
Not all patients in the Netherlands will be treated with intentionally curative treatment with surgery 
in the participating PLCRC hospitals, and some patients treated in PLCRC hospitals do not participate 
in the PLCRC. The proportion of patients that are eligible for adjuvant chemotherapy, and are 
included in participating PLCRC centres, is set at 40%. With these figures, we estimate that 595 
patients each year will be available to screen in 25 participating PLCRC hospitals. The prevalence of 
detectable ctDNA in these patients is 15%. Approximately 89.25 patients each year are expected to 
be eligible for inclusion and accrual will be completed somewhere between the third and fourth year 
of the study (accounting for start-up difficulties, we estimate that 70 patients each year will be 
included over the total inclusion period of three years).  
 
Calculation: 3500*0.5*0.85*0.4*0.15=89.25 patients/year. 206/89.25 = 2.31 years. 
 
With a detection rate of 15% for ctDNA after surgery in high-risk rectal cancer, 1373 patients’ blood 
samples need to be analysed for ctDNA to include all patients with detectable ctDNA (n=206), 103 
patients with detectable ctDNA for each group. The hypothesis of the REACT study is that adjuvant 
chemotherapy will improve disease-free survival of rectal cancer patients with detectable ctDNA 
after surgery and neoadjuvant treatment. Based on this hypothesis, patients with postoperative 
detectable ctDNA will be randomized to an experimental group receiving adjuvant chemotherapy, 
and a control group receiving standard of care (no additional treatment). 
 
The largest study discussed in this literature review (Tie et al.) found that the 3-year disease-free 
survival in patients with detectable ctDNA after surgery is 33% (95% CI: 16% to 72%). (28)  However, 
it has to be taken into account that 58% of these patients received adjuvant chemotherapy, and that 
the proportion of patients that would be disease-free is likely to be smaller if no additional treatment 
was administered. In the REACT population, patients with a pathological complete response will not 
be included, hereby excluding a group of patients with a relatively good prognosis.  
 
Based on these figures and considerations, we expect that the 2-year disease-free survival in patients 
a) with detectable ctDNA after surgery and neoadjuvant treatment, b) without a complete response, 
and c) without any additional treatment is 30%. We aim to demonstrate a hazard ratio of 0.55 for 
adjuvant chemotherapy (pure treatment effect), but that this hazard ratio will be reduced to 0.64 
when taken into account that 25% of patients in the intervention group will refuse experimental 
treatment or are not eligible for adjuvant chemotherapy due to comorbidity. This will translate into 
an improved disease-free survival in the adjuvant chemotherapy group from 30% to 55% (absolute 
risk increase of 25%).  
 
For the detection of a hazard ratio of 0.64 in the experimental arm, 30% 2-year disease-free survival 
in the control group, and with two-sided significance level α = 0.05 and 80% power, the number of 
patients needed to include is 206 in total (103 vs. 103).  
Every hospital should screen approximately 14 patients per year and include 2-3 patients with 
detectable ctDNA per year for adjuvant chemotherapy. 
 
 
 

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14.6 Planned analysis 
 
14.6.1 Analysis primary endpoint 
The endpoint for the primary analysis will be disease free survival. The formal test for 
difference in disease-free survival between the two groups will be done with a Cox regression 
analysis with adjustment for patient-, pre-treatment- and tumour characteristics. The actuarial 
method of Kaplan and Meier will be used to estimate disease-free survival probabilities at 
appropriate time points, while the Greenwood estimate will be used to construct 
corresponding 95% confidence intervals (CIs). Kaplan-Meier curves will be generated to 
illustrate disease-free survival, for all patients as well as by group. The final analyses will not be 
performed until the required numbers of events (118) have been observed, and the data have 
been validated.  
 
Primary endpoint of the study will be disease-free survival (DFS), calculated from the date of 
randomisation to the date of progression (recurrence) or death from any cause of the patient, 
whichever occurs first. Patients who did not experienced a recurrence and who are still alive at 
the end of the study will be censored at the date of last contact. 
 
14.6.2 Analysis secondary endpoint(s)  
In addition to intention-to-treat analysis to estimate the effect size, also a per protocol analysis 
(for pure treatment effect) will be carried out for disease-free survival and overall survival. This 
analysis will compare all (eligible) patients in the control group with those patients in the 
experimental group who actually started the adjuvant chemotherapy. 
Secondary endpoint of the study will be overall survival, calculated from the date of surgery to 
the date death from any cause of the patient. Patients who are still alive at the end of the 
study will be censored at the date of last contact. The disease-free survival and overall survival 
of ctDNA negative patients will also be collected and analysed to confirm the robustness of 
post-operative ctDNA as biomarker in rectal cancer. Survival outcomes will be compared 
between patients who had undetectable ctDNA versus patients who had detectable ctDNA and 
did not undergo any additional therapy (control group of the randomised patients). Quality of 
life questionnaires are presented at different time points. Comparison between both groups 
will be calculated by a random effects regression model and will be based on the intention to 
treat principle. Repeated measurement analysis will be used to evaluate within and between 
group differences. The results of the ctDNA analysis of the blood samples taken at the first 
follow-up of the patients who were ctDNA positive after surgery will be compared between 
the patients who received adjuvant chemotherapy and those who did not.  Within the group of 
patients with detectable ctDNA after surgery, the proportion of patients with detectable 
ctDNA in the peripheral blood at the timing of detection of recurrent disease on imaging will 
be assessed. 
 
14.6.3 Analysis other study parameters/endpoints 
In an exploratory analysis we will investigate the mutational profile and level of ctDNA of the 
ctDNA positive patients, the specific mutations that are demonstrated and possible correla-
tions with clinical and pathological parameters and time to recurrence. In patients of whom 
tumour tissue is available, we will compare mutational status of ctDNA and tumour tissue.  
 

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14.7 Interim analysis 
No interim analysis will be planned.  
 
14.8 (Statistical) criteria for termination of the trial 
While no interim analysis is planned, the trial may be terminated prematurely if certain unexpected 
circumstances arise that impede the successful completion of the study. Examples include inade-
quate patient screenings rates, changes in daily clinical practice due to newly published study results 
or other external factors that could compromise the ethical or scientific validity of the trial.   
 
14.9 Procedure for accounting for missing, unused and spurious data 
All the collected data are expected to be used, with exception of the data of patients that are 
considered ineligible afterwards based on information that should have been available at timing of 
ctDNA analysis.  
 
14.10 Procedure for reporting any deviation(s) from the original statistical plan 
Any deviation(s) from the original statistical plan will be described and justified in the protocol 
and/or in the final report, as appropriate.   
 
15. ETHICAL CONSIDERATIONS 
 
15.1 Declaration of Helsinki 
The study will be conducted according to the principles of the Declaration of Helsinki (75th WMA 
General Assembly, Helsinki, Finland, October 2024) and in accordance with the Medical Research 
Involving Human Subjects Act (WMO) the Good Clinical Practice (GCP)  guidelines, and this study 
protocol. 
15.2 Recruitment and informed consent procedures 
If patients have given written informed consent for PLCRC, and for withdrawal of additional blood 
samples and future (interventional) studies, after surgery and meeting the inclusion criteria, blood 
samples can be taken and analysed for the presence of ctDNA. Patients with detectable ctDNA can be 
randomised to the observational control group and the experimental group. Patients in the 
experimental group will be contacted by their treating physician (surgeon) that is included in the 
PLCRC study team. Patients will be asked to be contacted about the REACT study by one of the REACT 
investigators, for additional information about the REACT study. Patients in the control group will not 
be contacted, because they are anonymous for the study team and their treating physician.  
 
Patients are free to refuse to undergo the experimental intervention at all times, when offered. None 
of the patients that participate in this project will be withheld treatments that have been proven to 
be effective in their disease subgroup, without prior obtaining explicit informed consent of the 
patient.    
 
Information about REACT will be provided 2-4 weeks after ctDNA analysis in eligible patients. 
Patients will visit their treating physician (surgeon) at that time for a standard follow-up visit after 
surgery and when they fulfil criteria for REACT, they are randomised before this visit. The treating 
physician or research nurse will give information about REACT to patients randomised to the 
experimental group and about detectable ctDNA. 

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Patients who are interested in receiving adjuvant chemotherapy will be referred to the medical 
oncology department for chemotherapy counselling by a medical oncologist at the outpatient clinic. 
Additional information about REACT will be given by the REACT study team. 
Patients will then decide based on the given counselling and information about ctDNA analysis, risk 
of recurrence and harms and benefits of adjuvant chemotherapy, if they want to receive adjuvant 
chemotherapy.  
Patients will have time for consideration of participation and if they are willing to participate, they 
will sign informed consent for the REACT study and the schedule for adjuvant chemotherapy will be 
discussed with their treating physician. The formal written informed consent must be obtained 
before the start of adjuvant chemotherapy. 
 
Trials within cohorts (TwiCs) design 
In PLCRC (MEC-2016-369), patients are separately asked to give informed consent to be offered 
experimental interventions, and that their data can be used in comparative research. Although, at 
the time informed consent is signed, neither the patient nor the researcher knows for which 
(possibly running) studies this particular patient is or might become eligible. This is a situation of 
‘broad’ consent (patients do not know for which research questions their data will be used at time of 
signing consent), most predominant in biobank studies. However, this approach within the TwiCs is 
specifically chosen to deliver the least (psychological or emotional) distress to the patient, since they 
are only informed if their data will be used for research questions and if they still have a choice if 
they want to undergo the offered experimental intervention or not. Thus, this broad consent 
approach could be explained to protect the patient from experiencing distress in case they enter the 
control group without receiving the opportunity to influence their treatment course or the way in 
which the data will be used. Nevertheless, all patients are well informed that they can withdraw from 
the study (including the broad consent) at any time without reasoning. 
 
From the group of patients in the PLCRC cohort eligible for REACT the TwiCs randomly selects 
patients who will be offered the intervention. Patients from that same sub-cohort, who are not 
randomly selected, will undergo standard treatment (i.e. regular follow-up), without being notified 
about the fact that they were not (but could have been) randomly selected. All patients, randomly 
selected or not, are aware of the fact that they could have been invited for new treatments, and that 
their data are being used for evaluation of effectiveness of new interventions. The TwiCs is designed 
to prevent patients from receiving information about interventions they will not receive. We do 
acknowledge that some patients are therefore withheld from information about a treatment under 
study that some patients, comparable to themselves, will be offered to undergo, while they are not. 
This ‘patient-centred’ approach makes sure that informed consent for a particular intervention is 
sought only from those offered that intervention. Patients allocated to the control group are 
deliberately not informed about the presence of ctDNA, as this would entail an unnecessary burden 
for patients and their treating physicians. Furthermore, it might be uneasy for treating physicians to 
not offer any additional treatment, and could possibly result in drop-outs or even cross-over to the 
treatment group. 
 
 
 
 

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15.3 Benefits and risks assessment, group relatedness 
It is expected that this study will contribute to more personalized treatment and improved survival of 
rectal cancer patients with detectable ctDNA postoperatively and improved quality of care. 
The 103 patients in the experimental group that have detectable ctDNA after surgery are exposed to 
the risks of adjuvant chemotherapy. These risks are well-known from current clinical practice and 
patients will be counselled. The risk of additional blood withdrawals is negligible. 
 
Minors will not be included in this study. Patients who are unable to understand the information and 
give informed consent will not be included in the study. 
 
15.4 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the WMO. 
  
The sponsor (also) has an insurance which is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO). This insurance provides cover for damage to research subjects through 
injury or death caused by the study. 
 
The insurance applies to the damage that becomes apparent during the study or within 4 years after 
the end of the study. 
 
15.5 Compensation for subjects 
Patients that have detectable ctDNA after surgery and that are randomised to the experimental 
group, will be offered adjuvant chemotherapy. By participation in this study, these patients are in-
formed about their high risk of recurrence of disease and will be offered a treatment they would not 
have been offered in current clinical practice. The current data about the high recurrence rate in pa-
tients with detectable ctDNA will likely contribute to the preparedness of patients to receive adju-
vant chemotherapy. No financial incentives are provided to participants of the study, but travel costs 
can be compensated 
 
15.6 Compensation for investigators 
Not applicable. 
 
15.7 Other ethical considerations  
Not applicable.  
 
16. ADMINISTRATIVE ASPECTS, MONITORING AND CONFIDENTIALITY 
The study will be conducted in compliance with the protocol, with Clinical Trials Regulation No 
536/2014 and with the principles of good clinical practice. 
 
16.1 Approval initial application and substantial modifications 
The trial protocol, informed consent form, subject information leaflet, investigational medicinal 
product dossier, investigators brochure and any other documents required by the Regulation will be 
submitted for the regulatory approval before the clinical trial is started via CTIS. 
 

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The sponsor will also submit and obtain approval for substantial modifications to the original 
approved documents via CTIS. 
 
A ‘substantial modification’ is defined in the CTR as any change to any aspect of the clinical trial 
which is made after notification of a decision referred to in Articles 8, 14, 19, 20 or 23 and which is 
likely to have a substantial impact on the safety or rights of the subjects or on the reliability and 
robustness of the data generated in the clinical trial. 
 
16.2 Monitoring 
Monitoring will be done following the requirements of the Netherlands Federation of University 
Medical Centres (NFU) based on the ICH Good Clinical Practice guidelines. The REACT study is classi-
fied as a medicinal study with negligible risk. For every site there will be an initiating visit, at least two 
on study visits (one per year) and a closing out visit. The timing of visits will depend on inclusion 
speed, amount of included subjects and previously observed deviations. 
 
 
16.3 Recording, handling and storage of information 
 
16.3.1 Handling of data and data protection 
Individual patient information obtained as a result of this study is considered confidential 
and is handled conform the Dutch Act on Implementation of the General Data Protection 
Regulation; in Dutch: Uitvoeringswet AVG (UAVG) . Disclosure to third parties is prohibited, 
except for authorities involved with monitoring and quality control of the clinical study such 
as monitors, auditors, members of the Medical Research Ethics Committee and the health 
care inspectorate. Patients confidentiality will be ensured by using identification numbers 
(for example: EMC-001, EMC-002, etc.). A subject identification code list is safeguarded by 
the local principal investigator in case data needs to be linked back to an individual subject. 
Research data that can be traced to individual persons can only be viewed by authorized 
personnel. 
PLCRC data handling and storage will be conducted within the PLCRC study in UMC Utrecht, 
and supplemented with additional data by the IKNL Clinical Trial Centre. A separate REACT 
database (managed in Castor) is created for study-specific data, and will be filled in with the 
collected (pseudonymised) data from PLCRC and IKNL. The data will be kept for at least 25 
years. 
 
16.3.2 Source documents and case report forms (CRF) 
Source documents for this study will include hospital records and procedure reports and data 
collection forms. These documents will be used to enter data on the CRFs. Data reported on 
the CRF that are derived from source documents must be consistent with the source 
documents or the discrepancies must be explained. 
 
All documents will be stored safely in confidential conditions. On all study-specific 
documents other than the signed consent, the subject will be referred to by the study subject 
identification code. 
 
 

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16.3.3 Clinical trial master file and data archiving 
The sponsor and the investigator shall keep a clinical trial master file. The clinical trial master 
file shall at all times contain the essential documents relating to the clinical trial which allow 
verification of the conduct of a clinical trial and the quality of the data generated (CTR: Article 
57). 
 
The sponsor and the investigator shall archive the content of the clinical trial master file for 
at least 25 years after the end of the clinical trial, unless other EU law requires archiving for a 
longer period. The medical files of subjects shall be archived in accordance with national law 
(CTR: Article 58). 
 
The content of the clinical trial master file shall be archived in a way that ensures that it is 
readily available and accessible, upon request (CTR: Article 57). 
 
16.3.4 Collection and storage of biological samples 
Additional blood will be collected from consenting patients at the time of a regular blood draw 
for clinical practice. The blood samples will be coded with both the PLCRC PICNIC number and 
the REACT study-number. The PLCRC study team manages the patient information of all 
subjects and can identify the patient information of the biological sample if needed. 
Laboratory personnel will handle samples in a blinded fashion. Samples will be processed 
within 96 hours as described and stored at -80 degrees (plasma) or -30 degrees (cfDNA) until 
further analysis.  All blood samples will be stored for 25 years, unless the patient explicitly 
declines consent for this in the patient informed consent form. 
 
16.4 Audits and inspections and direct access to source data/documents 
This trial may be subject to internal or external monitoring, auditing or inspections procedure to 
ensure adherence to GCP. Access to all trial-related documents including direct access to source data 
will be given at that time. 
 
16.5 Reporting of serious breaches 
The sponsor will notify the Member States concerned about a serious breach of the Regulation or of 
the version of the protocol applicable at the time of the breach through CTIS without undue delay 
but not later than seven days of becoming aware of that breach (CTR: Article 52). 
 
16.6 Notification of the start and the end of the recruitment 
The sponsor will notify within 15 days each Member State concerned of the start of a clinical trial in 
relation to that Member State through CTIS (CTR: Article 36(1)). 
 
The sponsor will notify within 15 days each Member State concerned of the first visit of the first 
subject in relation to that Member State through CTIS (CTR: Article 36(2)). 
 
The sponsor will notify within 15 days each Member State concerned of the end of the recruitment 
of subjects for a clinical trial in that Member State through the EU (CTR: Article 36(3)). 

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16.7 Temporary halt/(early) termination 
The sponsor will notify within 15 days each Member State concerned of the end of a clinical trial in 
relation to that Member State through CTIS (CTR: Article 37(1)). 
The sponsor will notify within 15 days each Member State concerned of the end of a clinical trial in all 
Member States concerned and in all third countries in which the clinical trial has been conducted 
through CTIS (CTR: Article 37(3)). 
 
16.7.1 Temporary halt/early termination for reasons not affecting the benefit-risk 
balance 
The sponsor will notify with 15 days each Member State concerned of a temporary halt of a 
clinical trial in all Member States concerned for reasons not affecting the benefit-risk balance 
through CTIS (CTR: Article 37(5)). 
 
When a temporarily halted clinical trial for reasons not affecting the benefit-risk balance is 
resumed the sponsor will notify each Member State concerned through CTIS (CTR: Article 
37(6)). 
 
The sponsor will notify to the EU portal CTIS of early termination of the clinical trial for reasons 
not affecting the benefit-risk balance through CTIS. The reasons for such action and, when 
appropriate, follow-up measures for the subjects will be provided as well (CTR: Article 37(7)). 
 
16.7.2 Temporary halt/early termination for reasons of subject safety 
In accordance to article 38 of the CTR, the sponsor will suspend the study if there is sufficient 
ground that continuation of the study will jeopardise subject health or safety. The temporary 
halt or early termination of a clinical trial for reasons of a change of the benefit-risk balance 
will be notified to the Member States concerned through the EU portal CTIS without undue 
delay but not later than in 15 days of the date of the temporary halt or early termination. It 
shall include the reasons for such action and specify follow-up measures. The restart of the 
clinical trial following a temporary halt as referred to in paragraph 1 shall be deemed to be a 
substantial modification subject to the authorisation procedure laid down in Chapter III of the 
CTR (CTR: Article 38).  
 
16.8 Summary of the results 
Within one year from the end of a clinical trial in all Member States concerned, the sponsor will 
submit to the EU database CTIS a summary of the results of the clinical trial. The content of the 
summary of the results is set out in CTR Annex IV. It shall be accompanied by a summary written in a 
manner that is understandable to laypersons. The content of the summary is set out in CTR Annex V 
(CTR: Article 37(4)). 
 
16.9 Public disclosure and publication policy 
This study will be registered before start of accrual in the WHO recognised ClinicalTrials.gov 
(https://clinicaltrials.gov/). Results will be reported according to the CONSORT guidelines. Publica-
tions resulting from this study will be submitted to peer-reviewed journals. The principle investigator 
and study coordinators will prepare the manuscript together with those who substantially contrib-
uted to the study, at the discretion of the principle investigator. Any publication, abstract or preser-
vation based on patients included in this study must be approved by the primary investigator and the 

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study coordinators. This is applicable to any individual patient registered in the trial, or any subgroup 
of the trial patients. Such a publication cannot include any comparisons between the randomised 
groups or an analysis of any of the study end-points unless the final results of the trial have already 
been published. Within one year after the primary endpoint has been reached, final results of the 
study will be presented at (inter)national congresses and submitted to an international peer-re-
viewed journal.  
 
 
 

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