Smart Measurement of Circulating Tumor DNA: a Tumor-agnostic Computational Tool to Improve Colorectal Cancer Care
Phase: NA
Conditions: Colorectal Neoplasms, Colorectal Neoplasms Malignant, Colorectal Neoplasms, Hereditary Nonpolyposis, Hereditary Nonpolyposis Colon Cancer
Sponsor: Erasmus Medical Center

NL87476.078.24/ SMART 
 
 
 
 
 
 
 
TITLE: ‘Smart measurement of circulating tumor DNA: a tumor-agnostic computational tool 
to improve colorectal cancer care’ 
 
Short title 
The SMART study 
Version 
1.3 
Date 
15-4-2025 
Principal investigator(s)/project 
leaders 
1. Dr. Anja Wagner 
Dept. of Clinical Genetics, Room Ee2022 
Erasmus MC, Rotterdam, the Netherlands 
a.wagner@erasmusmc.nl / +31(0)641516016 
2. Dr. Saskia Wilting 
Dept. of Medical Oncology, Room Be 
Erasmus MC, Rotterdam, the Netherlands 
s.wilting@erasmusmc.nl / +31(0)612417667 
3. Prof. dr. Manon Spaander 
Dept. of Gastroenterology, Room Na-616 
Erasmus MC, Rotterdam, the Netherlands 
Sponsor 
Dept. of Medical Oncology and Clinical Genetics, 
Erasmus MC Cancer Institute, Rotterdam, the 
Netherlands 
Subsidising party 
Dutch Cancer Society (KWF Kankerbestrijding) 
Nr. 14976 

NL87476.078.24/ SMART 
 
 
 
 
 
 
Independent expert (s) 
J.M.C. Collee 
Dept. of Clinical Genetics, Room Ee2024 
Erasmus MC, Rotterdam, the Netherlands 
j.collee@erasmusmc.nl +31(0)629217116 
Laboratory sites 
Erasmus MC – Cancer Institute 
Afdeling Interne Oncologie 
Lab. Translational Cancer Genomics 
Gebouw-kamer; BE-410 
Dr. Molewaterplein 40 
3015 GD Rotterdam 

NL87476.078.24/ SMART 
 
 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Head of Department: 
1. Prof. Dr. Henk Verheul 
Dept. of Medical Oncology 
Erasmus MC, Rotterdam, the Netherlands 
h.verheul@erasmusmc.nl 
 
2. Prof. Dr. Tjitske Kleefstra 
Dept. of Clinical Genetics 
Erasmus MC, Rotterdam,  the Netherlands 
t.kleefstra@erasmusmc.nl 
 
 
 
 
 
 
 
 
Sept. 18th 2024 
Principal Investigators: 
 
1. Dr. Anja Wagner 
Dept. of Clinical Genetics, Room Ee2022 
Erasmus MC, Rotterdam, the Netherlands 
a.wagner@erasmusmc.nl / 
+31(0)641516016 
 
2. Dr. Saskia Wilting 
Dept. of Medical Oncology, Room Be 
Erasmus MC, Rotterdam, the Netherlands 
s.wilting@erasmusmc.nl / 
+31(0)612417667 
 
3. Prof. Dr. Manon Spaander 
Dept. of Gastroenterology, Erasmus MC, 
Rotterdam, the Netherlands 
v.spaander@erasmusmc.nl /  
+31 (0) 653417029 
 
 
 
 
 
 
 
 
 
Sept 11, 2024 
 
 
 
16-09-2024 
Collaborating Investigators 
1. Prof. Dr. Monique van Leerdam 
Dept. of Gastroenterology, Leiden 
 
 
 

NL87476.078.24/ SMART 
 
 
 
University Medical Centre, Leiden, the 
Netherlands 
m.e.van_leerdam@lumc.nl  
2. Maartje Nielsen 
Dept. of Clinical Genetics, Leiden 
University Medical Centre, Leiden, the 
Netherlands 
m.nielsen@lumc.nl 
 
 
10-9-2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE .............................................................................. 10 
1.1 
Lynch Syndrome ..................................................................................................... 10 
1.2 
Preventing cancer through surveillance programs ................................................... 10 
1.3 
ctDNA analysis in cancer detection ......................................................................... 10 
1.4 
Cell-free DNA based surveillance for LS-associated cancers .................................. 11 
2. 
OBJECTIVES ................................................................................................................ 13 
3. 
STUDY DESIGN ............................................................................................................ 14 
3.1 
Patient inclusion ..................................................................................................... 14 
3.2 
Sample collection ................................................................................................... 14 
3.3 
cfDNA methylation and sWGS analysis .................................................................. 14 
3.4 
Workflow ................................................................................................................ 15 
3.5 Timetable .................................................................................................................... 17 
3.5 
Population (base) ................................................................................................... 19 
3.6 
Inclusion criteria ...................................................................................................... 19 
3.7 
Exclusion criteria..................................................................................................... 19 
3.8 
Sample size calculation .......................................................................................... 20 
5. TREATMENT OF SUBJECTS........................................................................................... 20 
6. 
INVESTIGATIONAL PRODUCT .................................................................................... 20 
7. 
NON-INVESTIGATIONAL PRODUCT ........................................................................... 20 
8. 
METHODS .................................................................................................................... 21 
8.1 
Study parameters/endpoints ................................................................................... 21 
8.1.1 
Main study parameter/endpoint ........................................................................ 21 
8.1.2 
Other study parameters ................................................................................... 21 
8.2 
Randomisation, blinding and treatment allocation ................................................... 21 
Not applicable. .................................................................................................................. 21 
8.3 
Study procedures ................................................................................................... 21 
8.3.1 
Inclusion of participants ................................................................................... 21 
8.3.2 
Blood collection ............................................................................................... 21 
8.3.3 
ctDNA analysis ................................................................................................ 22 
8.4 
Withdrawal of individual subjects ............................................................................ 22 
8.4.1 
Specific criteria for withdrawal (if applicable) .................................................... 22 
Not applicable. ............................................................................................................... 22 
8.5 
Replacement of individual subjects after withdrawal ................................................ 22 
Not applicable. .................................................................................................................. 22 
8.6 
Follow-up of subjects withdrawn from treatment ...................................................... 22 
Not applicable. .................................................................................................................. 22 
8.7 
Premature termination of the study ......................................................................... 22 
Not applicable. .................................................................................................................. 22 
9. 
SAFETY REPORTING................................................................................................... 23 
9.1 
Temporary halt for reasons of subject safety ........................................................... 23 
9.2 
AEs, SAEs and SUSARs ........................................................................................ 23 

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9.2.1 
Adverse events (AEs) ...................................................................................... 23 
9.2.2 
Serious adverse events (SAEs) ....................................................................... 23 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .......................... 24 
Not applicable. ............................................................................................................... 24 
9.3 
Annual safety report ............................................................................................... 24 
Not applicable. .................................................................................................................. 24 
9.4 
Follow-up of adverse events ................................................................................... 24 
9.5 
[Data Safety Monitoring Board (DSMB) / Safety Committee] ................................... 24 
Not applicable. .................................................................................................................. 24 
10. 
STATISTICAL ANALYSIS .......................................................................................... 25 
10.1 Primary study parameter(s)..................................................................................... 25 
10.2 Secondary study parameter(s) ................................................................................ 25 
10.3 Other study parameters .......................................................................................... 26 
10.4 Interim analysis (if applicable) ................................................................................. 26 
Not applicable. .................................................................................................................. 26 
11. 
ETHICAL CONSIDERATIONS ................................................................................... 27 
11.1 Regulation statement .............................................................................................. 27 
11.2 Recruitment and consent ........................................................................................ 27 
11.3 Objection by minors or incapacitated subjects (if applicable) ................................... 27 
Not applicable ................................................................................................................... 27 
11.4 Benefits and risks assessment, group relatedness .................................................. 27 
11.5 Compensation for injury .......................................................................................... 28 
11.6 Incentives ............................................................................................................... 28 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .......................... 29 
12.1 Handling and storage of data and documents ......................................................... 29 
12.2 Monitoring and Quality Assurance .......................................................................... 29 
12.3 Amendments .......................................................................................................... 30 
12.4 Annual progress report ........................................................................................... 30 
12.5 Temporary halt and (prematurely) end of study report ............................................. 30 
12.6 Public disclosure and publication policy .................................................................. 30 
13. 
STRUCTURED RISK ANALYSIS ............................................................................... 31 
14. 
REFERENCES .......................................................................................................... 32 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS 
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; 
in Dutch: Algemeen Beoordelings- en Registratieformulier (ABR- 
formulier) 
AE 
Adverse Event 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
 
cfDNA 
ctDNA 
CNV 
CRC 
CV 
DNA 
DSMB 
EC 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
Cell-free DNA 
Copy Number Variation 
Circulating Tumor DNA 
Colorectal Cancer 
Curriculum Vitae 
Deoxyribonucleic Acid 
Data Safety Monitoring Board 
Endometrial Cancer 
EU 
European Union 
EudraCT European drug regulatory affairs Clinical Trials 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product 
IMPD 
LS 
LUMC 
METC 
 
MMR 
MRD 
MSI 
NKI 
PIF 
Investigational Medicinal Product Dossier 
Lynch Syndrome 
Leiden University Medial Centre 
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
Mismatch Repair 
Minimal Residual Disease 
Microsatellite Instability 
Dutch Cancer Institute (in Dutch: Nederlands Kanker Instituut) 
Patient Information Form 

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(S)AE 
SDR 
SDV 
SNV 
 
SPC 
Sponsor 
 
 
 
 
 
 
StOET 
 
SUSAR 
sWGS 
TMF 
(Serious) Adverse Event 
Source Data Review 
Source Document Verification 
Single Nucleotide Variant 
Summary of Product Characteristics; in Dutch: officiële 
productinformatie IB1-tekst 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
Dutch Foundation for Detecting Hereditary Tumors (in Dutch: Stichting 
Opsporing Erfelijke Tumoren) 
Suspected Unexpected Serious Adverse Reaction 
Shallow Whole Genome Sequencing 
Trial Master File 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; 
in Dutch: Uitvoeringswet AVG 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch- 
wetenschappelijk Onderzoek met Mensen 

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SUMMARY 
 
 
Rationale: Lynch Syndrome carriers have a predisposition to develop various types of 
cancer, especially colorectal cancer (CRC) and endometrial cancer (EC). LS patients are 
advised to undergo surveillance by colonoscopy every 2 year and gynaecological 
surveillance. This surveillance is deemed burdensome and fails to detect a small part of the 
developing CRCs and the majority of extra-colonic cancers. To ensure prevention and early 
detection of cancer, a reliable and accessible test is needed. 
Recent studies have shown the potential of the detection of tumor-derived DNA fragments 
(circulating tumor DNA; ctDNA). Various molecular characteristics can be used to 
discriminate ctDNA from healthy circulating cell-free DNA. Current ctDNA assays with the 
highest sensitivity and specificity to detect for example minimal residual disease (MRD) after 
surgery are mostly tumor-informed, which means prior information is needed from the tumor 
tissue about the molecular alterations present. As this information is not available for the 
detection of newly arising tumors, the aim of this study is to evaluate the use of an optimized 
combination of tumor-agnostic ctDNA characteristics for the detection of newly developing 
tumors. 
Objective: To evaluate the feasability of a new tumor-agnostic ctDNA assay, the ctDNA 
estimator, to detect newly developing tumors in Lynch carriers. 
Study design: case-control study. 
Study population: For validation of the ctDNA estimator, we will include: (1) LS carriers, 
whom at time of inclusion, have a newly diagnosed tumor and have not received any therapy 
yet. (2) Samples from the control population (LS carriers without a tumor diagnosis), that 
have already been collected according to the CATCA study (METC:2019-0119). 
Intervention: LS carriers will be asked to participate at time of a new cancer diagnosis. After 
informed consent is given, blood (3 x 10 mL) will be obtained for ctDNA analysis to compare 
the ctDNA estimates to that in the control population. 
Main study parameters/endpoints: The agreement between estimated ctDNA fractions and 
the presence of cancer. 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: Blood will be drawn (3 x 10 mL) from LS carriers with a recently 
diagnosed cancer, which will be combined with a routine hospital visit when possible. The 
risk of blood collection by venepuncture is negligible. 

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1. INTRODUCTION AND RATIONALE 
1.1 Lynch Syndrome 
Over 15,000 individuals are diagnosed with colorectal cancer (CRC) in the Netherlands each 
year, of which 3-5% can be attributed to Lynch Syndrome (LS) [1]. LS is a genetic 
predisposition for various types of cancer next to early-onset CRC, including cancer of the 
endometrium, ovaries, small bowel, stomach, urothelium, biliary tract, pancreas, brain and 
skin [2]. It is caused by a germline mutation in one of the DNA mismatch repair (MMR) 
genes; MLH1, MSH2, MSH6, PMS2, or in the 3’end of the EpCAM gene [3-7]. Consequently, 
microsatellite instability (MSI-H) is a hallmark of LS associated tumors [8]. 
In the Netherlands in all newly diagnosed CRC and endometrial cancer under the age of 70 
immunohistochemical staining of the MMR proteins is performed to evaluate the 
microsatellite status of the tumor and improve LS diagnosis [9]. Overall, about 10% of all 
CRC display an MSI-H phenotype, of which about a third is due to LS [10]. LS inherits in an 
autosomal dominant way. Once a germline mutations in MSH2, MSH6, PMS2, or in the 3’end 
of the EpCAM gene is detected in a patient, presymptomatic testing is available to at risk 
relatives and carriers are offered colorectal and/or gynecological surveillance to decrease 
their risk of cancer [11]. 
 
1.2 Preventing cancer through surveillance programs 
LS carriers have a lifetime risk up to 70% to develop CRC, and female carriers additionally 
have a lifetime risk of up to 60% to develop EC [12]. Due to these high risks, LS carriers are 
advised to undergo biennial colonoscopy from 25-35 years of age – and females additional 
yearly gynecological surveillance between 40 and 60 years of age – to reduce this cancer 
risk. However, colonoscopies and endometrial screening are burdensome to the patient and 
fail to detect part of the newly developing tumors [13, 14]. Furthermore, the use of 
surveillance programs for other LS-associated tumors especially tumors of the ovaries, small 
bowel, stomach, urothelium, biliary tract, pancreas, brain and skin remain controversial, as 
no added value to the detection of carcinomas has been shown [15]. 
 
1.3 ctDNA analysis in cancer detection 
Cell-free DNA (cfDNA) is shed into the circulation via apoptotic and necrotic cells, which 
means it can contain a fraction of circulating tumor DNA (ctDNA) in case a tumor is present 
in the body. CRC has a particularly high shedding rate of tumor material into the bloodstream 
[16]. This provides a promising future for minimally invasive assays for diagnosis, aiming at 
detection of tumor-derived circulating DNA fragments (ctDNA) in the blood [17]. Recent 
results show that the detection of minimal residual disease (MRD) - using ctDNA in stage II 

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CRC patients following surgical removal of the tumor – can be used to reduce the number of 
patients treated with adjuvant chemotherapy, without negatively impacting the clinical 
outcome [18]. However, current assays to detect MRD often use tumor-informed designs. 
This means that patient-specific assays are developed, based on available genetic 
information of the tumor tissue to maximize sensitivity and specificity for this assay. However, 
this assay design is complicated and expensive, as sequencing of tumor tissue is needed in 
combination with development of patient-specific assays that need to be tested. This is a 
time-consuming process and impedes implementation into the clinical setting. Additionally, 
tumor-informed assays cannot be used to detect de novo tumors, as no prior information 
about the molecular landscape is available in this setting. 
Based on aforementioned information, there is an unmet need for a sensitive and specific 
assay to detect ctDNA in individuals, without the need for prior information from the tumor 
tissue (i.e. tumor-agnostic). The implementation of such an assay would simplify MRD 
detection after surgery and may improve current surveillance programs as well. 
 
1.4 Cell-free DNA based surveillance for LS-associated cancers 
To detect ctDNA in the total pool of cfDNA, numerous tests can be performed. This includes 
assessment of chromosomal copy number variations (CNVs), microsatellite instability (MSI), 
single nucleotide variants (SNVs), and DNA methylation assays. The presence of ctDNA is 
often determined by a combination of aforementioned assays and can be used in various 
settings [19]. Recent technological advances underline the promise of certain tumor-agnostic 
(i.e. without prior tissue-based information) ways to detect ctDNA. Schrag et al. have 
demonstrated the feasibility of tumor-agnostic assays, which can predict both presence of a 
tumor and its origin [20]. Circulating cfDNA methylation analyses for both early detection and 
classification of various cancer types have been proven useful [21, 22]. Two recent studies 
show this potential of cfDNA methylation profiling to detect CRC at an early stage [23, 24]. 
The recently developed field of ‘fragmentomics’ (i.e. the investigation of fragmentation 
patterns in cfDNA) shows promise for sensitive detection of cancer. Circulating DNA 
fragments of cancer patients have a differential size profile compared to cfDNA fragments in 
healthy individuals. This difference can be used in assays to increase the sensitivity of 
chromosomal copy number profiling in cfDNA [25]. Additionally, these differential cfDNA 
fragmentomic profiles can directly distinguish cancer patients from healthy individuals with 
high sensitivity and specificity [25, 26]. Lo et al. have shown that these fragmentomic 
features can be retrieved via various paired-end sequencing methods, including methylation 
sequencing [27]. Features from different molecular read-outs (e.g. methylation and copy 
number profiles) from both cfDNA methylation profiling and fragmentomics have a 

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complementary value [28, 29]. Combining cfDNA methylation profiling and fragmentomics 
will capture information of the circulating tumor genome on a broader spectrum, permitting a 
higher analytical sensitivity for the detection of minute traces of ctDNA. 
 
Current literature indicates that cfDNA methylation profiling, fragment size-enriched 
chromosomal copy number profiling, and fragmentomics – or combinations of these – may 
have the potential to provide a tumor-agnostic assay for sensitive detection of newly 
developing tumors. Recently we showed that cfDNA methylation profiles could discriminate 
microsatellite instable advanced colorectal tumors from microsatellite stable tumors [30]. 
Here, we propose to evaluate the potential of a new ctDNA estimator to detect newly 
developing tumors and to improve current surveillance programs for LS carriers. 

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2. OBJECTIVES 
 
 
Primary objective: 
This project aims to deliver a tumor-agnostic ctDNA estimator and provide proof-of-concept 
of its value for early diagnosis of new colorectal tumors in high-risk individuals. 
 
Secondary objective(s): 
Obtain proof of principle that the ctDNA estimator can also play a role in the early detection 
of newly developing non-colorectal tumors in LS carriers. 

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3. STUDY DESIGN 
This is a monocenter (with potential to be multi-center) case-control study in which we will 
test the ability of the constructed ctDNA estimator to detect newly developing tumors. We 
will do this by comparing cfDNA data extracted from already collected blood from LS 
carriers without a newly developed tumor (controls) to that from LS carriers with a newly 
developed tumor, whom have not had any form of therapy yet (cases). 
 
 
3.1 Patient inclusion 
Due to their high risk for CRC cancer, LS carriers are under regular surveillance. LS carriers 
with a newly diagnosed cancer will be asked for informed consent in the period between the 
diagnosis and treatment. 
Also, patients suspected to have Lynch based on routinely performed immunohistochemical 
staining of the MMR proteins on their newly diagnosed tumor will be invited to participate in 
the study. MSI is a molecular hallmark of Lynch-associated tumors. About half of patients 
with a suspect LS tumor will have underlying LS. 
 
3.2 Sample collection 
If informed consent is given, blood (3 x 10 mL) will be collected for this study. Blood will be 
drawn via venipuncture. If possible, this will be combined with routine blood sampling prior to 
treatment. Blood will be collected in blood-stabilizing tubes and sent to the laboratory of 
Translational Cancer Genomics at the Medical Oncology Department. Plasma will be 
processed within 96 hours and stored at -80 degrees according to standard protocol. Blood 
from controls is readily available at the Erasmus MC from the previous CATCA study 
(METC:2019-0119). 
Cell-free DNA will be isolated from 2-4 mL of plasma using the qiaAMP circulating cell-free 
DNA kit (Qiagen). The obtained cell-free DNA will be quantified with the Quant-iT PicoGreen 
dsDNA Assay Kit (Thermo Fisher Scientific). In addition, we will evaluate the ratio of cell-free 
DNA (cfDNA) to higher molecular weight genomic DNA using the ProNex DNA QC assay 
(Promega). 
 
3.3 cfDNA methylation and sWGS analysis 
cfDNA will be isolated out of plasma using the QiaAmp circulating nucleic acid kit (Qiagen) 
according to the manufacturer’s instructions. This will be done for the samples of both the 50 
cases and 50 controls included in this study. Ten ng of this isolated cfDNA will be analyzed 
via MeD-seq, whereas another 5-10 ng cfDNA will be analysed via sWGS. 

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The required features for the cfDNA estimator will be extracted from the data, using various 
in-house or publicly available pipelines for MeD-seq and sWGS data. 
For DNA methylation profiling we will use the MeD-seq assay [31] , which was shown to have 
important advantages over other genome-wide methylation assays, such as high CpG 
coverage (~50% of CpGs in the genome), low costs, no conversion of the DNA, and no 
dependency on DNA-binding antibodies. Therefore, this assay is ideally suited for the 
profiling of small amounts of DNA and we have recently shown that we can indeed use the 
MeD-seq assay to yield reliable cfDNA methylation profiles [32] . 
For fragmentomics analyses, we will use 5-10 ng of cfDNA to directly generate sequencing 
libraries without fragmenting the cfDNA. Libraries will be quantified using the Tapestation 
(Agilent) and pooled equimolarly. The resulting pool will be sequenced on the NextSeq 500 
system (Illumina) generating 150 bp paired end reads. In total we will aim to generate >10M 
reads per sample (>0.5x coverage). The resulting data will be analyzed with in house 
sequencing data pipelines to obtain a range of fragmentomic features. In short, adapters and 
indexes will be trimmed from the reads and the trimmed reads will be mapped to the 
GRCh38 human genome assembly using burrows-wheeler alignment [33] 
 
Mapped data will be used to extract: 
1) ctDNA-enriched chromosomal copy number profiles, which will be summarized into an 
tumourfraction estimate using IchorCNA [34] 
2) genome-wide fragmentation patterns 
3) cfDNA fragment size distributions 
4) the fraction of fragments derived from open and closed chromatin 
5) the diversity of the fragment ends, which will be summarized into a single metric by first 
calculating a normalized Shannon entropy and subsequently combining this with the 
proportions of 29 selected trinucleotide fragment-endings into a recently developed FrEIA 
score [35]. 
 
The independently developed multi-omic ctDNA estimator will be used to estimate the ctDNA 
fraction estimates in cases (i.e. LS carriers with a tumor) and controls (i.e. LS patients 
without a tumor). The sensitivity and specificity of the ctDNA estimator will be determined in 
these 50 cases and 50 controls. 
 
3.4 Workflow 
The workflow of this study is depicted in figure 1 and 2. In case a tumor is detected in a 
(suspect) LS carrier, consent to be contacted for further information on the cfDNA estimator 

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study by the researcher is asked by the collaborating physician and written information on 
the study is supplied to the patient. Informed consent is asked during a live or telephone 
consult by the researcher and blood drawing is organized in the interval between cancer 
diagnosis and therapy. As blood from 50 healthy LS carriers (controls) is readily available, 
we only need to collect blood samples of 50 LS carriers with a newly diagnosed tumor 
(cases). cfDNA will be isolated from both cases and controls, and the previously described 
assays will be performed. After data is obtained, the independently developed multimodal 
ctDNA estimator will be used and its sensitivity and specificity will be determined in our case- 
control setting. 
 
 
 
 
 
Figure 1. Flowchart for patient inclusion and sample collection. 

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Figure 2. Workflow for sample and data collection and analysis. 
 
 
 
3.5 Timetable 
This project will be carried out in 4 years by a dedicated PhD student. Techniques and 
methods used for this study are already up and running. In the first year focus is on preparing 
the study (e.g. developing patient information folders, obtaining METC approvals, other 
administrative work), followed by the start of patient inclusion. We estimate we will reach our 
goal of 50 patient inclusions in the third year. Synchronously to patient inclusion, blood 
sampling will be carried out, along with the necessary pre-processing to plasma. We expect 
our data to be complete and ready for analysis by the end of the third year, after which the 
ctDNA estimator will be evaluated. The results will be presented at (inter)national meetings 
and will be published in peer-reviewed, open-access scientific journals. 

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Figure 3. Gantt chart depicting the timeline of this study. 

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4. STUDY POPULATION 
4.1 Population (base) 
We will include individuals with molecularly proven Lynch Syndrome. In total, we need to 
analyze 50 cases (as specified in the power calculation below). Based on the StOET (the 
Dutch Foundation for detection of Hereditary Tumors) database, we expect that an average 
of 70 LS carriers under surveillance are diagnosed with an LS-associated tumor per year in 
the Netherlands. Apart from that, about half of newly diagnosed MSI-H CRC and EC cases 
suspect for LS will turn out to be LS-associated by following diagnostic testing. These are 
about 15 newly diagnosed LS carriers with a tumor at our center per year. Including also 
these patients will facilitate the inclusion of 50 cases within the inclusion time. 
Next to our own regional LS-task force, participation of the clinical geneticists and 
gastroenterologists with expertise on LS in the LUMC, and if needed the other Dutch 
academic centers, will be asked to participate. To ensure sufficient recruitment, patient 
awareness will also be created via the Lynch Polyposis foundation. 
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
- 
(suspect) LS carriers who: 
o Have proven Lynch Syndrome (MMR gene or EpCAM mutation), or have a proven 
MSI-H tumor; 
o Are ≥ 18 years of age; 
o Have been diagnosed with any form of cancer at time of inclusion, but have 
had no treatment yet; 
o Have granted informed consent to participate in this study. 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
- 
Case (suspect) LS carriers who: 
o Are unwilling to undergo extra blood sampling; 
o Are <18 years; 
o Have no newly diagnosed tumors at time of inclusion; 
o Have been treated for their tumor at time of inclusion; 
o Not able to read or understand Dutch language or mentally not capable. 

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4.4 Sample size calculation 
This study is a case control study in order to test the ctDNA estimator in a relatively small 
cohort of high-risk carriers. Since most surveillance colonoscopies performed every two 
years in LS carriers turn out negative and ctDNA estimator could be performed yearly we 
judge the ctDNA estimator to be successful if it detects at least 60% of all tumors at a 
maximal false-positive rate of 25% in healthy carriers. With a power of 80% and at an alpha 
of 0.05, we need to include at least 36 carriers with a tumor and 36 healthy carriers, 
according to the Chi-squared statistic with continuity correction to compare proportions with a 
dichotomous outcome between two samples. To ensure sufficient individuals with good 
quality data and adequate follow-up, we therefore propose to include 50 carriers with and 50 
carriers without a tumor. In order to obtain sufficient LS carriers with a newly diagnosed 
tumor we expect to include an additional 30-50 suspect LS carriers, who will not be analysed 
for this study, since the diagnosis LS is not confirmed. 
 
5. TREATMENT OF SUBJECTS 
Not applicable. 
 
6. INVESTIGATIONAL PRODUCT 
Not applicable. 
 
 
7. NON-INVESTIGATIONAL PRODUCT 
Not applicable. 

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8. METHODS 
8.1 Study parameters/endpoints 
 
8.1.1 Main study parameter/endpoint 
The main study endpoint is to evaluate the ability of an independently developed ctDNA 
estimator to detect newly developing tumors in LS carriers. Sensitivity and specificity of this 
new detection method will be established in a case-control set-up where we aim to detect 
at least 60% of all tumors at a maximal false-positive rate of 25% in healthy carriers. 
 
8.1.2 
Other study parameters 
 
These data are needed as background information to interpret results of the molecular 
analyses: 
- 
Demographics: 
o Age; 
o Gender; 
o Genetic predisposition (i.e. which MMR gene mutation); 
- 
Comorbidities; 
- 
Detected malignancies and their characteristics, such as: size, differentiation, grade, 
and stage. 
 
 
8.2 Randomisation, blinding and treatment allocation 
Not applicable. 
 
 
8.3 Study procedures 
An overview of assessments for (suspect) LS carriers in this study is depicted in 
figures 1 and 2. After informed consent is obtained, baseline characteristics and 
information on comorbidity will be collected from the patient’s medical file. 
 
 
8.3.1 
Inclusion of participants 
LS carriers under surveillance and suspect LS patients based on their tumor profile will be 
informed about this study at moment of cancer diagnosis by their physician and given an 
informative letter. When the patient gives permission to be contacted, the researcher will 
contact the patient for further information within one week. Informed consent will be asked for 
participation in this study and for blood sampling. Blood sampling will be scheduled, 

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preferably in combination with routine sampling for the patient’s cancer treatment, giving the 
patient enough time to (re)consider participation. In case of suspect LS, the department of 
Clinical Genetics will communicate the subsequent DNA results from genetic testing. Based 
on these results, patients with proven LS will be analyzed. 
8.3.2 
Blood collection 
As per protocol, blood will be sampled before start of treatment, regardless of tumor type. 
After consent is given for collection and storage of blood for further molecular analysis, extra 
blood sampling (3 x 10 mL) will be ordered preferably at their routine blood-sampling 
appointment before therapy. This is to minimize extra burden of an additional hospital visit for 
this blood sampling. Blood will be collected in cell stabilizing blood collection tubes and send 
to in the laboratory of Translational Cancer Genomics at the Medical Oncology department. 
8.3.3 
ctDNA analysis 
Molecular analyses in cfDNA/ctDNA obtained via blood will be performed according to 
already established protocols within the Erasmus MC. These analyses do not interfere with 
routine diagnostics. If participants give consent, unused plasma will be stored for 15 years 
for possible related and/or subsequent studies. 
 
8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without 
any consequences. The investigator can decide to withdraw a subject from the study 
for urgent medical reasons. 
8.4.1 Specific criteria for withdrawal (if applicable) 
 
Not applicable. 
 
 
8.5 Replacement of individual subjects after withdrawal 
Not applicable. 
 
 
8.6 Follow-up of subjects withdrawn from treatment 
Not applicable. 
 
 
8.7 Premature termination of the study 
Premature termination of the study is not likely. 

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if 
there is sufficient ground that continuation of the study will jeopardise subject health or 
safety. The sponsor will notify the accredited METC without undue delay of a temporary halt 
including the reason for such an action. The study will be suspended pending a further 
positive decision by the accredited METC. The investigator will take care that all subjects are 
kept informed. 
 
9.2 AEs, SAEs and SUSARs 
 
9.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the 
study, whether or not considered related to trial procedure. Adverse events will not be 
recorded, because the risk of adverse events after blood withdrawal by venepuncture is 
negligible. 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that 
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention but could have been based upon 
appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
 
Serious adverse events will not be recorded, because the risk of serious adverse events after 
blood withdrawal by venepuncture is negligible. 

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9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
 
Not applicable. 
 
 
9.3 Annual safety report 
Not applicable. 
 
 
9.4 Follow-up of adverse events 
Adverse events will be handled as usual practice, according to standard of care. 
 
 
9.5 Data Safety Monitoring Board (DSMB) / Safety Committee 
Not applicable. 

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10. STATISTICAL ANALYSIS 
10.1 Primary study parameter(s) 
The primary objective of this study is to provide proof-of-principle that the blood-based ctDNA 
estimator is able to detect newly developing tumors in LS carriers. The primary study 
parameter is therefore the distinction between ctDNA estimates between LS carriers with and 
without cancer, according to the ctDNA estimator. This is determined by analyzing total 
cfDNA of both cases and controls, as described in chapter 3. Per sample a ctDNA estimate 
will be calculated (value between 0-1). This resulting value will be compared between the 
cases and controls with a Mann-Whitney U test. These calculations will be done by using R 
(version 4.4.1). 
As this is a proof-of-principle study, the focus of analysis will be on correctly detecting ctDNA 
in cases and controls. An ROC-curve will be constructed on the basis of the test-
performance and evaluated for possible clinical applications. Further optimization of the test 
regarding sensitivity and specificity will need to be done in a bigger prospective cohort study. 
Also, the optimization of sensitivity and/or specificity depends on how this test could be 
implemented. Likely, the test will be an addition to the normal colonoscopy surveillance, thus 
higher specificity would be preferred. 
 
10.2 Secondary study parameter(s) 
As a secondary objective we focus on expanding the ctDNA estimator beyond colorectal cancer 
diagnostics. ctDNA analyses will incorporate several parameters to distinguish tissue-of-origin from 
cfDNA/ctDNA fragments. These analyses will include, for example, differential-methylated regions 
(DMRs), which are tissue/cell-specific.

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10.3 Other study parameters 
Baseline patient characteristics are needed to interpret the results of the molecular analyses. 
These characteristics include: 
- 
Demographics: 
o Age; 
o Gender; 
o Genetic predisposition (i.e. which MMR gene mutation); 
- 
Comorbidities; 
- 
Detected malignancies and their characteristics, such as: size, differentiation, grade, 
and stage. 
Demographics are needed to match the cases to controls, to ensure baseline characteristics 
are evenly distributed. Information on (possible) comorbidities are needed to interpret cfDNA 
analyses, as there may be DNA abnormalities that are not related to the tumor. Tumor 
characteristics are also needed to interpret cfDNA analyses and relate the outcomes of the 
analyses to the tumor status. If this tumor-data is not present within the Erasmus MC, data 
and material will be collected via PALGA. 
 
 
10.4 Interim analysis (if applicable) 
Not applicable. 

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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(version of 2024) and in accordance with the Medical Research Involving Human Subjects 
Act (WMO). 
 
11.2 Recruitment and consent 
Eligible patients, being (suspect) LS carriers, will be informed by their physician about this 
study at moment of cancer diagnosis, given an informative letter and asked permission to be 
contacted by the researcher for further information. In a live or telephone contact, the 
researcher asks informed consent for participation in this study. Upon consent extra blood 
sampling will be organized. Patients will follow routine diagnostic work-up and treatment 
according to their cancer diagnosis, thus time between cancer diagnosis and subsequent 
blood sampling may vary. Regardless of the duration of this period, participants are free to 
(re)consider their participation during this time. Furthermore, patients can approach involved 
researchers/clinicians or the independent expert to ask questions any time prior or during 
participation. Patients have the right to withdraw their consent at any time during the study. 
 
11.3 Objection by minors or incapacitated subjects (if applicable) 
Not applicable 
 
 
11.4 Benefits and risks assessment, group relatedness 
As surveillance programs for LS are considered burdensome and adenomas and CRC are 
sometimes missed [36], we aim to develop a more sensitive and less invasive tool for the 
detection of newly developing tumors in high-risk individuals. 
We believe there are no relevant risks associated with participation. It is necessary for 
evaluation of this ctDNA estimator that blood is collected for further molecular work-up and 
analysis. Blood will be collected (3x 10ml) via routine venepuncture, which we do not 
consider as a large burden to patients, especially since we aim to combine it with already 
scheduled blood drawing needed for their treatment. As cancer diagnosis precedes 
participation, the cfDNA analysis will be based on information from the tumor. Thus, there is 
no extra burden of unforeseen health problems or unprecedented findings.

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11.5 Compensation for injury 
We believe participation in this study is without any risks. 
 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of the 
WMO. 
 
11.6 Incentives 
Not applicable. 

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
Participant data will be stored in a password-protected database according to the General 
Data Protection Regulation (in Dutch: ‘Algemene Verordening Gegevensbescherming’, 
AVG). All patients will be asked informed consent for processing and usage of personal data 
(additional to asking informed consent to participate in the study). This database will be 
secured with a password and safeguarded by the investigator. Individuals are given a study 
identification number and personal information will be stored separately from the study data. 
This study will be reported to the Privacy Knowledge Office of the Erasmus MC, to be 
included in the Register of Processing Operations (in Dutch: verwerkingsregister). Patients 
will be informed about the handling of their personal records; additionally, they are provided 
with the contact details of the Data Protection Officer of the Erasmus MC (dhr. P. Van 
Hoogdalem), the controller (in Dutch: verwerkingsverantwoordelijke, dr. A. Wagner ) and 
where to find additional information regarding the rights of the data subjects (Privacy 
Knowledge Office, Erasmus MC). Data and human material used in this study will be stored 
for 15 years for possible related and/or subsequent studies. Human material that is not used 
by the end of the study will be destroyed, unless patients grant informed consent for storage 
and use in future studies. 
 
12.1 Monitoring and Quality Assurance 
Monitoring of the study will be done via the Research Agency Sophia (Onderzoeksbureau 
Sophia). This will be done yearly, as in compliance with the WMO guidelines. The following 
will be monitored: 
- 
Patient Information Forms (PIFs) 
- 
Informed Consent Procedure 
- 
Compliance with in- and exclusion criteria 
- 
Qualifications PI and team 
- 
Safety 
- 
Compliance with research procedures 
- 
Collection and storage of samples 
- 
Site File (TMF) 
- 
Source Data Review (SDR) 
- 
Source Document Verification (SDV) 
- 
SAEs 

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12.2 Amendments 
Amendments are changes made to the research after a favourable opinion by the accredited 
METC has been given. All amendments will be notified to the METC that gave a favourable 
opinion. 
 
12.3 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accredited 
METC once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial, serious 
adverse events/ serious adverse reactions, other problems, and amendments. 
 
12.4 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last patient’s last visit. 
The sponsor will notify the METC immediately of a temporary halt of the study, including the 
reason of such an action. 
 
In case the study is ended prematurely, the sponsor will notify the accredited METC within 15 
days, including the reasons for the premature termination. 
 
Within one year after the end of the study, the investigator/sponsor will submit a final study 
report with the results of the study, including any publications/abstracts of the study, to the 
accredited METC. 
 
12.5 Public disclosure and publication policy 
When the study has ended, the principal investigator will be responsible for the public 
disclosure and publication of study outcome(s). Results will be presented on (inter)national 
professional meetings/symposia and submitted to journals for publication. 

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13. STRUCTURED RISK ANALYSIS 
Not applicable. 

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