Prospective, Multicenter, Comparative, Paired Study to Validate a microRNA-based Fecal Test for Colorectal Cancer Screening. The miRFec Study
Phase: NA
Conditions: Colorectal Cancer
Sponsor: Fundacion Clinic per a la Recerca Biomédica

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 1 of 40 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prospective, multicenter, comparative, paired study to validate a 
microRNA-based fecal test for colorectal cancer screening, surveillance 
and diagnosis. The miRFec study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol code: miRFec001 
 
Protocol version and date: version 5 (February 5th, 2025) 
 
Promoter: Fundació Clínic per a la Recerca Biomèdica (Clinic Foundation for Biomedical 
Research) 
 
Coordinator and principal investigator: Dr. Antoni Castells. Gastroenterology 
Department, Hospital Clínic of Barcelona 
 
 
 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 2 of 40 
1. SUMMARY 
 
1.1 Title:  
Prospective, multicenter, comparative, paired study to validate a microRNA-based 
fecal test for colorectal cancer screening, surveillance and diagnosis. The miRFec 
study. 
1.2 Promoter:  
Fundació Clínic per a la Recerca Biomédica (Clinic Foundation for Biomedical 
Research). 
1.3 Protocol code: 
miRFec001 
1.4 Coordinator and principal investigator:  
Dr. Antoni Castells  
Gastroenterology Department 
Hospital Clínic of Barcelona 
Villarroel 170 
08036 Barcelona 
Catalonia, Spain 
Phone number: +34 93 227 57 03 
E-mail: castells@clinic.cat  
1.5 Researchers and centers where the study is planned to be performed: 
▪ Hospital Clinic of Barcelona, Spain - Dr. Antoni Castells 
▪ Hospital del Mar, Barcelona, Spain - Dr. Xavier Bessa 
▪ Hospital General de Alicante, Spain - Dr. Rodrigo Jover 
▪ Hospital de Ourense, Spain - Dr. Joaquín Cubiella 
▪ Hospital Universitario de Canarias, Spain - Dr. Goretti Hernández 
▪ Hospital de Donostia, Spain - Dr. Luis Bujanda 
▪ National Research Institute of Oncology, Warsaw, Poland - Dr. Michael Kaminiski 
and Dr. Joanna Nowakowska  
▪  
▪ Beskid Oncology Center - Municipal Hospital, Bielsko-Biała, Poland – Dr. 
Sławomir Kozieł 
1.6 Ethics Committee (CEIm): 
Hospital Clínic of Barcelona, Spain. 
1.7 Population included: 
Study population will include 2700 individuals in stage I and 2690 individuals in stage 
II: 
▪ Male and female aged 50 to 75 (average-risk) referred to colonoscopy-based 
colorectal cancer (CRC) screening. 
▪ Male and female aged 18 or older with family history of CRC (moderate-risk) 
referred to colonoscopy-based CRC screening. 
▪ Male and female aged 18 or older with personal history of colorectal polyps referred 
to colonoscopy surveillance. 
▪ Male and female aged 18 or older with personal history of CRC referred to 
colonoscopy surveillance. 
▪ Male and female aged 18 or older with colorectal symptoms referred to diagnostic 
colonoscopy. 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 3 of 40 
1.8 Study design and phase:  
Prospective, multicenter, comparative, paired, in vitro diagnostic (IVD) clinical 
performance study. 
1.9 Primary endpoint: 
To compare the performance (i.e. sensitivity) of the miRFec test with respect to fecal 
immunochemical testing (FIT) for the detection of advanced colorectal neoplasm (i.e. 
CRC, advanced adenomas [AA] or advanced serrated lesions [ASL]). 
1.10 Secondary endpoints: 
▪ To compare the specificity of the miRFec test with respect to FIT. 
▪ To compare the sensitivity of the miRFec test with respect to FIT for the 
detection of CRC. 
▪ To compare the sensitivity of the miRFec test with respect to FIT for the 
detection of AA. 
▪ To compare the sensitivity of the miRFec test with respect to FIT for the 
detection of ASL. 
▪ To compare the detection rate for advanced colorectal neoplasm of the miRFec 
test with respect to FIT. 
▪ To compare discrimination capacity of the miRFec score and fecal hemoglobin 
concentration for the detection of advanced colorectal neoplasm. 
▪ To compare the discrimination capacity of the miRFec score and fecal 
hemoglobin concentration for the detection of CRC. 
▪ To compare the cost-effectiveness of the miRFec test with respect to FIT for 
the detection of advanced colorectal neoplasm. 
1.11 Main variable 
Sensitivity for the detection of advanced colorectal neoplasm (i.e. CRC, AA or ASL). 
1.12 Secondary variables 
▪ Specificity 
▪ Sensitivity for CRC, AA and ASL detection 
▪ Detection rate for advanced colorectal neoplasm 
▪ Discrimination capacity for advanced colorectal neoplasm and CRC detection 
▪ Cost-effectiveness for advanced colorectal neoplasm detection 
1.13 Inclusion criteria 
▪ Male or female aged 50 to 75 (average-risk) referred to colonoscopy-based CRC 
screening. 
▪ Male or female aged 30 to 80 with family history of CRC (moderate-risk) referred to 
colonoscopy-based CRC screening. 
▪ Male and female aged 18 or older with personal history of colorectal polyps referred 
to colonoscopy surveillance. 
▪ Male and female aged 18 or older with personal history of CRC referred to 
colonoscopy surveillance. 
▪ Male and female aged 18 or older with colorectal symptoms referred to diagnostic 
colonoscopy. 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 4 of 40 
1.14 Exclusion criteria 
▪ Lack of informed consent to participate 
▪ Personal history of Lynch syndrome 
▪ Personal history of adenomatous or hamartomatous polyposis 
▪ Personal history of serrated polyposis syndrome 
▪ Personal history of inflammatory bowel disease 
▪ Personal history of total colectomy for any reason 
▪ Family history of Lynch syndrome 
▪ Family history of adenomatous or hamartomatous polyposis 
▪ Family history of serrated polyposis syndrome 
1.15 Follow-up and timeline 
▪ Stage I: threshold search (February 2022 – April 2024) 
▪ Stage II: performance evaluation (February 2025 – December 2027) 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 5 of 40 
 
2. ABBREVIATIONS 
 
AA 
 
Advanced adenoma 
AE 
 
Adverse event  
ASL 
 
Advanced serrated lesion 
BBPS  
Boston bowel preparation scale 
CRC  
Colorectal cancer 
e-CRF 
Electronic case report form 
FIT 
 
Fecal immunochemical test  
miRNA 
Micro RNA  
IVD 
 
In vitro diagnostic 
SAE  
Serious adverse event 
SAP  
Statistical analysis plan 
SAR  
Serious adverse reaction  
SPS  
Serrated polyposis syndrome 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 6 of 40 
3. INDEX 
 
1. SUMMARY ...................................................................................................................... 2 
2. ABBREVIATIONS ............................................................................................................ 5 
3. INDEX .............................................................................................................................. 6 
4. GENERAL INFORMATION .............................................................................................. 8 
4.1. Study identification .................................................................................................... 8 
4.2. Promoter identification ............................................................................................... 8 
4.3. Coordinator and principal investigator ....................................................................... 8 
5. JUSTIFICATION .............................................................................................................. 9 
6. PURPOSE OF THE STUDY .......................................................................................... 11 
6.1. Hypothesis .............................................................................................................. 11 
6.2. Primary endpoint ..................................................................................................... 11 
6.3. Secondary endpoints ............................................................................................... 11 
6.4. Main and secondary variables ................................................................................. 11 
7. STUDY DESIGN ............................................................................................................ 12 
7.1. Study population...................................................................................................... 12 
7.2. Study duration ......................................................................................................... 12 
7.3. End of study ............................................................................................................ 12 
8. PARTICIPANTS ............................................................................................................. 13 
8.1. Inclusion criteria ...................................................................................................... 13 
8.2. Exclusion criteria ..................................................................................................... 13 
8.3. Withdrawal criteria ................................................................................................... 13 
9. PROCEDURES ............................................................................................................. 15 
9.1. Recruitment ............................................................................................................. 15 
9.2. Fecal sample collection ........................................................................................... 15 
9.3. Endoscopy and pathology reporting ........................................................................ 15 
9.4. Sample processing .................................................................................................. 16 
9.5. Definitions ............................................................................................................... 16 
9.5.1. Adenomatous polyposis .................................................................................... 16 
9.5.2. Hamartomatous polyposis ................................................................................ 16 
9.5.3. Serrated polyposis syndrome ........................................................................... 16 
9.5.4. Lynch syndrome ............................................................................................... 16 
9.5.5. Invasive colorectal cancer ................................................................................ 17 
9.5.6. Advanced adenoma .......................................................................................... 17 
9.5.7. Advanced serrated lesion ................................................................................. 17 
9.5.8. Advanced colorectal neoplasm ......................................................................... 17 
10. STUDY CALENDAR .................................................................................................... 18 
11. SAFETY ASSESSMENT ............................................................................................. 20 
11.1. Safety parameters definitions ................................................................................ 20 
11.2. Safety parameters evaluation ................................................................................ 20 
11.3. Register of adverse events .................................................................................... 20 
12. STATISTICS ................................................................................................................ 21 
12.1. Sample size calculation ......................................................................................... 21 
12.2. Statistical analysis plan (SAP) ............................................................................... 21 
13. ETHICS AND LEGAL ASPECTS ................................................................................. 23 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 7 of 40 
14. DATA MANAGEMENT, CONFIDENTIALITY AND REGISTER FILE ........................... 24 
14.1. Confidentiality ........................................................................................................ 24 
14.2. Electronic case report form (e-CRF) ...................................................................... 25 
14.3. Data transfer.......................................................................................................... 26 
14.4. Source documents ................................................................................................ 26 
14.5. Responsibilities ..................................................................................................... 26 
15. BIOLOGICAL SAMPLES MANAGEMENT ................................................................... 27 
16. FUNDING .................................................................................................................... 28 
17. PUBLICATION POLICY ............................................................................................... 29 
18. REFERENCES ............................................................................................................ 30 
ANNEX 1. Information sheet .............................................................................................. 32 
ANNEX 2. Participant consent form ................................................................................... 37 
ANNEX 3. Instructions for fecal sample collection ............................................................. 38 
ANNEX 4. Staging according to American Joint Committee on Cancer (8th version) ......... 39 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 8 of 40 
4. GENERAL INFORMATION 
 
4.1. Study identification 
▪ Title:  Prospective, multicenter, comparative, paired study to validate a microRNA-
based fecal test for colorectal cancer screening, surveillance and diagnosis. The 
miRFec study. 
▪ Protocol code: miRFec001 
▪ Version 1. Date: June 16th, 2021 
▪ Version 2. Date: January 28th, 2022 
▪ Version 3. Date: August 25th, 2023 
▪ Version 4. Date: February 23rd, 2024 
▪ Version 5. Data: February 5th, 2025 
 
4.2. Promoter identification 
 
Fundació Clínic per a la Recerca Biomèdica (Clinic Foundation for Biomedical 
Research) 
 
Rosselló 149-153 
 
08036 Barcelona 
 
Catalonia, Spain 
 
4.3. Coordinator and principal investigator 
 
Dr. Antoni Castells 
 
Gastroenterology Department 
 
Hospital Clínic of Barcelona 
 
Villarroel 170 
 
08036 Barcelona 
 
Catalonia, Spain 
 
Phone number: +34 93 227 57 03 
 
E-mail: castells@clinic.cat 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 9 of 40 
5. JUSTIFICATION 
Colorectal cancer (CRC) is the third most common cancer and the second leading 
cause of cancer-related death in Western countries, accounting for almost 1.8 million of new 
cases and 800.000 deaths in 20181. Although 90% of patients diagnosed at early stages 
have an overall survival of more than 5 years, this figure decreases to 10% in patients 
diagnosed at advanced stages with distant metastasis2. Interestingly, evidence from several 
studies has shown that CRC screening is effective and cost-effective in the average-risk 
population3.  
Recommended CRC screening strategies fall in two broad categories: stool tests 
(occult blood –guaiac test and fecal immunochemical test [FIT]– or exfoliated DNA tests) 
and structural exams (flexible sigmoidoscopy, colonoscopy and CT colonography)4. While 
structural exams detect both cancer and premalignant lesions, stool tests primarily identify 
cancer because of their limited sensitivity to detect advanced adenomas (AA)5. Moreover, 
in a two-step screening scenario, which is the most extended worldwide2, the relatively low 
specificity of the initial examination results in a high false-positive rate, leading to a 
significant number of unnecessary colonoscopies6.  
It is important to point out that to maximize the impact and ensure high coverage and 
equity of access, population-based organized screening programs are highly recommended, 
as opposed to case-finding or opportunistic screening, since they have an explicit policy and 
a team responsible for organization and invitation processes as well as to monitor and 
improve quality and outcomes7. In that context, almost all countries have chosen a two-step 
screening with FIT as the first step. It is important to note, however, that although the highest 
level of evidence supports the implementation of FIT-based screening, many countries have 
constrained their programs to manage the resultant colonoscopy workload in an equitable, 
timely, and high-quality manner2. For instance, countries such as England or Australia have 
initially constrained the age range of those offered screening, while others such as the 
Netherlands and Scotland have tailored the sensitivity of FIT –using a higher threshold– to 
reduce colonoscopy needs2. 
The use of biomarkers for screening purposes appears as an appealing approach to 
overcome the above-mentioned limitations8. MicroRNAs (miRNA) are small endogenous 
non-coding RNAs of 18 to 25 nucleotides that negatively regulate gene expression at post-
transcriptional level by either repressing transcript translation or inducing the degradation of 
target mRNAs9. It is important to highlight that they are involved in several biological 
processes, i.e. carcinogenesis10. Indeed, deregulated miRNAs reflect physiopathological 
states and allow distinguishing different stages and subtypes of cancer, including CRC, as 
well as to discriminating neoplastic patients from healthy individuals11. Interestingly, it seems 
that precursor lesions also show an altered miRNA pattern that could be, in part, secreted 
to the extracellular milieu. As a consequence, miRNAs detected in different body fluids, i.e. 
feces12, have been suggested as potential biomarkers for CRC screening. In that sense, in 
a recent study from our research group, we have technically and clinically validated a 
predictive algorithm based on a fecal miRNA signature –the miRFec test, a Gradient 
Boosting Machine-generated algorithm that includes two fecal miRNAs (miR-421 and miR-
27a-3p) and fecal hemoglobin concentration, along with age and gender– able to identify 
patients with advanced colorectal neoplasm more accurately than hemoglobin concentration 
in FIT-positive individuals13, 14.  
In the last years, the use of FIT has been proposed for other indications, besides CRC 
screening. Indeed, most population-based, organized screening programs have substituted 
colonoscopy surveillance by FIT in those patients in whom non-advanced colorectal lesions 
(i.e. tubular adenomas smaller than 10 mm with low-grade dysplasia and serrated polyps 
smaller than 10 mm without dysplasia) were detected. Moreover, it has been suggested that 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 10 of 40 
a similar surveillance strategy could also be applied to patients with advanced colorectal 
lesions based on the low recurrence rate after a complete, high-quality colonoscopy. Finally, 
FIT is also commonly used to triage those individuals with colorectal symptoms who may 
benefit from a diagnostic colonoscopy. All these strategies look forward to increasing 
effectiveness and cost-effectiveness of both screening, surveillance and diagnosis by 
reducing unnecessary colonoscopies. 
The present study aims to compare the effectiveness and cost-effectiveness of the 
miRFec test concerning FIT for the detection of advanced colorectal neoplasm among 
individuals participating in CRC screening, colorectal surveillance, and colorectal diagnosis. 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 11 of 40 
6. PURPOSE OF THE STUDY 
 
6.1. Hypothesis 
The miRFec test –a Gradient Boosting Machine-generated algorithm that includes two 
fecal miRNAs (miR-421 and miR-27a-3p) and fecal hemoglobin concentration, along with 
age and gender– is more effective and cost-effective than FIT for the identification of patients 
with advanced colorectal neoplasm (i.e. CRC, AA or advanced serrated lesions [ASL]) 
among individuals participating in CRC screening, colorectal surveillance, and colorectal 
diagnosis. 
 
6.2. Primary endpoint 
The main goal of the study is to compare the sensitivity of the miRFec test with respect 
to FIT for the detection of advanced colorectal neoplasm.  
 
6.3. Secondary endpoints 
▪ To compare the specificity of the miRFec test with respect to FIT. 
▪ To compare the sensitivity of the miRFec test with respect to FIT for the detection of 
CRC. 
▪ To compare the sensitivity of the miRFec test with respect to FIT for the detection of 
AA. 
▪ To compare the sensitivity of the miRFec test with respect to FIT for the detection of 
ASL. 
▪ To compare the detection rate for advanced colorectal neoplasm of the miRFec test 
with respect to FIT. 
▪ To compare the discrimination capacity of the miRFec score and fecal hemoglobin 
concentration for the detection of advanced colorectal neoplasm. 
▪ To compare the discrimination capacity of the miRFec score and fecal hemoglobin 
concentration for the detection of CRC. 
▪ To compare the cost-effectiveness of the miRFec test with respect to FIT for the detection of 
advanced colorectal neoplasm. 
 
6.4. Main and secondary variables 
▪ Main variable: Sensitivity for the detection of advanced colorectal neoplasm (i.e. 
CRC, AA or ASL). 
▪ Secondary variables: specificity; sensitivity for CRC, AA and ASL detection; detection 
rate for advanced colorectal neoplasm; discrimination capacity for advanced 
colorectal neoplasm and CRC detection; cost-effectiveness for advanced colorectal 
neoplasm detection. 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 12 of 40 
7. STUDY DESIGN 
Prospective, multicenter, comparative, paired, in vitro diagnostic (IVD) clinical 
performance study, carried out in medical centers of Spain and Poland.  
Eligible individuals will undergo two different examinations –miRFec test and FIT– in a 
unique stool sample collected at home. Results of both tests will be compared to those 
obtained in the colonoscopy (with pathology results when colorectal lesions were identified), 
which will be used as the reference method. 
 
7.1. Study population 
Study population will include 9,670 individuals: 
▪ Male and female aged 50 to 75 (average-risk) referred to colonoscopy-based 
colorectal cancer (CRC) screening. 
▪ Male and female aged 18 or older with family history of CRC (moderate-risk) 
referred to colonoscopy-based CRC screening. 
▪ Male and female aged 18 or older with personal history of colorectal polyps 
referred to colonoscopy surveillance. 
▪ Male and female aged 18 or older with personal history of CRC referred to 
colonoscopy surveillance. 
▪ Male and female aged 18 or older with colorectal symptoms referred to diagnostic 
colonoscopy. 
 
7.2. Study duration  
The study will last up to 6 years, which includes two consecutive stages: stage I 
(threshold search), aimed at establishing the most adequate threshold of miRFec test for 
advanced colorectal neoplasm detection, and stage II (performance evaluation), to compare 
the clinical performance of the miRFec test with respect to FIT. 
 
7.3. End of study 
The recruitment process will end up when completing the required sample size (2700 
individuals in stage I and 2690 in stage II). On the other hand, the study will finish when 
results of the miRFec test and FIT are correlated with colonoscopy findings (with pathology 
results when colorectal lesions were identified). 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 13 of 40 
8. PARTICIPANTS  
The study population will include individuals undergoing colonoscopy as primary 
screening approach in the context of both average and moderate-risk screening programs. 
 
8.1. Inclusion criteria 
▪ Male and female aged 50 to 75 years (average-risk) referred to colonoscopy-based 
CRC screening. This group includes individuals without family history of CRC as well 
as those with a family history limited to one first-degree relative with CRC diagnosed 
at the age of 50 or older, or any number of second- and third-degree relatives with 
CRC regardless the age of diagnosis.  
▪ Male or female aged 18 or older with family history of CRC (moderate-risk) referred 
to colonoscopy-based CRC screening. More specifically, this condition corresponds 
to individuals with two or more first-degree relatives with CRC regardless the age of 
diagnosis, or one first-degree relative with CRC diagnosed before the age of 50. 
▪ Male and female aged 18 or older with personal history of colorectal polyps (i.e. 
adenomas or serrated lesions) referred to colonoscopy surveillance. 
▪ Male and female aged 18 or older with personal history of CRC referred to 
colonoscopy surveillance. 
▪ Male and female aged 18 or older with colorectal symptoms (abdominal pain, 
bleeding and change in bowel habits, among others) referred to diagnostic 
colonoscopy. 
 
8.2. Exclusion criteria 
▪ Lack of informed consent to participate 
▪ Personal history of Lynch syndrome 
▪ Personal history of adenomatous or hamartomatous polyposis 
▪ Personal history of serrated polyposis syndrome 
▪ Personal history of inflammatory bowel disease 
▪ Personal history of total colectomy for any reason 
▪ Family history of Lynch syndrome 
▪ Family history of adenomatous or hamartomatous polyposis 
▪ Family history of serrated polyposis syndrome 
 
8.3. Withdrawal criteria 
According to Helsinki’s Declaration, all subjects participating in the study can withdraw 
whenever, without giving any reason and with no repercussion in their medical care. Any 
withdrawal from the study must be informed to the monitor of the study and documented in 
the e-CRF and participant’s medical record. Withdrawal criteria includes:  
▪ Exclusion criteria not reported at baseline visit  
▪ Participant does not deliver the fecal sample 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 14 of 40 
▪ Poor fecal sample quality (overfilling or insufficient amount)  
▪ Participant does not undergo colonoscopy 
▪ Inadequate bowel preparation in three consecutive colonoscopies. 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 15 of 40 
9. PROCEDURES 
 
9.1. Recruitment 
All individuals included in average-risk and moderate-risk CRC screening programs 
carried out in participating centers, those undergoing colorectal surveillance for previous 
lesions, and those with symptoms suggesting a colorectal disease are scheduled for a 
medical or nurse visit, in which they are asked for demographic data, personal and family 
history of cancer, comorbidities, usual medication, as well as any symptom of CRC. Once 
anamnesis is completed, a screening colonoscopy is scheduled. At this time, individuals will 
be offered to participate in the present study.  
All candidates to participate in the study will be explained orally about purpose of the 
study and given a written information sheet (Annex 1). Any doubt about the nature of study, 
benefits and potential risks will be solved. If the subject agrees to participate, informed 
consent will be signed (Annex 2). It will be informed about the freedom to revoke the 
informed consent without penalty or changes in clinical management, as well as the 
protection of their personal data and medical history according to current legislation. 
The recruitment will be competitive between all sites. 
 
9.2. Fecal sample collection 
Once the subject accepts to participate in the study, a fecal sample collection kit will 
be delivered together with the sample collection instructions (Annex 3). The sample must be 
collected at home before starting bowel preparation, more specifically between 24-72 hours 
before the day that colonoscopy is scheduled.  
Sample must be kept in the refrigerator (+4ºC) until the day of colonoscopy, when it 
must be brought and delivered to the endoscopy unit staff. 
 
9.3. Endoscopy and pathology reporting 
All colonoscopies will be performed by experienced endoscopists. Quality of 
colonoscopy will be ensured following guidelines of the European Society of Gastrointestinal 
Endoscopy (ESGE)15 and monitored.  
Colon cleansing will be scored according to the Boston bowel preparation scale 
(BBPS) (Table 1)16. In brief, right (i.e. cecum and ascending colon), transverse (i.e. hepatic 
flexure, transverse colon and splenic flexure) and left (i.e. descending colon, sigmoid and 
rectum) colon are scored from 0 to 3. These segments’ score is summed up for a total BBPS 
score. A punctuation of minimum 2 is required in each segment of the colon. Not achieving 
an adequate bowel preparation in three consecutive colonoscopies will be considered a 
withdrawal criterion of the study. 
Table 1. Boston bowel preparation scale 
Score 
Description 
0 
Unprepared colon segment with mucosa not seen because of solid stool that cannot be cleared 
1 
Portion of mucosa of the colon segment seen, but other areas of the colon segment are not well 
seen because of staining, residual stool and/or opaque liquid 
2 
Minor account of residual staining, small fragments of stool and/or opaque liquid, but mucosa of 
colon segment is seen well 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 16 of 40 
Score 
Description 
3 
Entire mucosa of colon segment seen well with no residual staining, small fragments of stool or 
opaque liquid 
 
All detected lesions must be described, documented and recorded in the electronic 
case report form (e-CRF). Decision of removing and/or taking biopsies of any detected 
lesion will correspond to the endoscopist. Similarly, other therapeutic decisions, including 
need for surgical resection, will correspond to the patient's responsible physician. 
Invasive cancer will be considered when malignant cells are observed beyond the 
muscularis mucosa, and graded according to the American Joint Committee on Cancer 
guidelines (8th version) (Annex 4)17. Carcinoma in situ (Tis, stage 0) will be considered 
equivalent to high-grade dysplasia and, therefore, this lesion will be classified as AA. 
 
9.4. Sample processing 
Both miRNA expression and FIT will be analyzed in a central laboratory (Hospital Clinic 
of Barcelona) according to previously published procedures13, 14. For this purpose, samples 
will be kept in a refrigerator (+4ºC) of the participating center until shipping to the central 
laboratory. 
 
9.5. Definitions 
 
9.5.1. Adenomatous polyposis 
▪ More than 100 adenomatous polyps (classical form) or 20-100 adenomatous polyps 
(attenuated form) in one or more colonoscopies (cumulative figure), or 
▪ Any number of adenomas in a gene mutation carrier in any causative gene (APC, 
MUTYH, POLE, POLD1, NTHL1) 
 
9.5.2. Hamartomatous polyposis 
 
Any number of hamartomatous polyps in a gene mutation carrier in any causative 
gene (SMAD4, BMPR1A, ENG, STK11, PTEN) 
 
9.5.3. Serrated polyposis syndrome 
According to World Health Organization (2019), serrated polyposis syndrome is 
defined as: 
▪ ≥5 serrated polyps proximal to the rectum, all being at least 5 mm in size, with ≥2 of 
them >10mm.  
▪ >20 serrated polyps of any size along the colon with ≥ 5 proximal to the rectum 
 
9.5.4. Lynch syndrome 
 
Gene mutation carrier in any DNA mismatch repair gene (MLH1, PMS2, MSH2, 
MSH6) 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 17 of 40 
9.5.5. Invasive colorectal cancer 
 
Invasive CRC will be considered when malignant cells are observed beyond the 
muscularis mucosa.  
 
9.5.6. Advanced adenoma 
 
Adenomas ≥10 mm in size, or with villous architecture, or high-grade dysplasia or 
intramucosal carcinoma (Tis) will be classified as AA. 
 
9.5.7. Advanced serrated lesion 
 
Serrated lesions ≥10 mm in size or with dysplasia will be classified as ASL. 
 
9.5.8. Advanced colorectal neoplasm 
 
Advanced colorectal neoplasm will be defined as CRC, AA or ASL.  
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 18 of 40 
10. STUDY CALENDAR 
 
Tasks 
Baseline 
visit 
Day -3 to -1 
Day 0 
(colonoscopy) 
Day 1 to 7 
Case 
completion 
Inclusion/exclusion 
criteria check 
 
 
 
 
 
Participant information 
sheet delivery 
 
 
 
 
 
Consent form signature 
 
 
 
 
Kit and fecal sample 
collection instructions 
delivery 
 
 
 
 
 
At home fecal sample 
collection 
 
 
 
 
 
Kit (fecal sample) return 
 
 
 
 
Colonoscopy 
 
 
 
 
Shipping samples to 
central laboratory 
 
 
 
 
 
Withdrawal criteria 
check 
 
 
 
 
 
Introduction of 
colonoscopy/pathology 
data into e-CRF 
 
 
 
 
 
 
▪ Baseline visit. Study investigators will ask for personal and family history, and check 
whether candidate fulfill inclusion/exclusion criteria to participate in the study. If that is 
the case, the subject will get a copy of the information sheet, will sign the consent form, 
and will be given a kit for fecal sample collection and instructions to collect the sample. 
▪ Day -3 to -1 (with respect the day of colonoscopy). The subject will collect a fecal 
sample at home, 24-72 hours before colonoscopy and always before starting laxatives 
(bowel preparation). Once the sample is collected, it must be kept in a refrigerator (+4ºC) 
until transporting it to the endoscopy unit. 
▪ Day 0 (colonoscopy). Patients will deliver the fecal sample to study investigators. 
Samples must be kept in a refrigerator (+4ºC) of the participating center until shipping to 
the central laboratory.  
▪ Day 1 to 7 (with respect to the day of colonoscopy). Samples will be shipped to the 
central laboratory once a week, ideally on Monday from Polish centers and Wednesday 
from Spanish centers.  

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 19 of 40 
▪ Case completion. When all information is available, data from colonoscopies, surgical 
procedures, and pathology (if lesions are detected) will be introduced in the e-CRF.  
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 20 of 40 
11. SAFETY ASSESSMENT 
The investigator is responsible for detecting and documenting any adverse event that 
fills in with the criteria and definition of severe adverse event that appear in this protocol. 
 
11.1. Safety parameters definitions 
▪ Adverse event (AE). Any harmful incident for a patient participating in a clinical trial 
using medicines, devices or medical procedures, even though there is no relation 
between the incident and the treatment used. 
▪ Serious adverse event (SAE) / Serious adverse reaction (SAR). An adverse event 
or adverse reaction is considered serious if it may cause death, require hospitalization 
or prolong the duration of it, cause invalidity or permanent or important disability, or 
cause a congenital malformation. Any suspicion of adverse event considered 
important in terms of medical point of view, will also be considered as serious and 
must be reported, even though they do not meet the criteria mentioned above. 
Additionally, medical staff must notify any suspicion of infectious agent transmission. 
"Life threat" is understood as a situation in which, in the opinion of physician, the 
patient was at a real risk of death at the time of the adverse event or the adverse 
reaction and, had there not been a timely therapeutic intervention, death would have 
occurred. It does not mean that the adverse event or adverse reaction hypothetically 
could have caused death if it had been more intense. The concept "require 
hospitalization" will exclude both planned hospitalizations for scheduled treatments 
and those that have been planned or anticipated before the start of study concerning 
a pre-existing medical situation. 
▪ Unexpected adverse reaction. It refers to an event or reaction that is not listed in 
the investigator’s brochure or is not listed at the specificity or severity that has been 
observed. 
 
11.2. Safety parameters evaluation 
Participation in this study will not represent any increased risk in addition to those 
associated with the colonoscopy already indicated for a screening purpose.  
For safety evaluation, SAE occurring up to 30 days after colonoscopy will be recorded 
in the e-CRF. The most frequent SAE include adverse reaction to sedation, bleeding and 
perforation if any lesion should be resected. 
 
11.3. Register of adverse events 
SAE and any defect of the device (i.e. fecal sample collection kit) will be recorded in 
the e-CRF. 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 21 of 40 
12. STATISTICS 
 
12.1. Sample size calculation 
This study includes two consecutive stages. The first one would consist of determining 
the optimal diagnostic threshold of the miRFec score in a population-based screening setting 
among naive participants (i.e. individuals who were not selected because of a FIT positive 
result, as it actually occurred in our previous study13, 14). The second stage aims at 
measuring the comparative performance of the miRFec test with respect to FIT. Therefore, 
sample size of this second stage depends upon estimates of the first stage and population 
characteristics.  
In order to validate the hypothesis of the miRFec test having greater performance (i.e. 
sensitivity) than FIT, sample size has been calculated to detect the estimated difference 
using a normal approximation, with a type I error of 0.025 (MH correction), a prevalence of 
CRC of 5‰, and type II error of 0.1. First simulations suggest a subject population of 3,600 
for stage I, and 6,070 for stage II (total, 9,670 individuals). However, sample size 
requirements may vary depending on advanced colorectal neoplasm prevalence and, 
consequently, sample size of stage I will be recalculated after collection of 2,700 samples, 
whereas sample size of stage II will be recalculated after stage I completion. 
Because of the observed difficulty to complete stage I recruitment at previously 
established time point, we have launched a scientific collaboration with the Netherlands 
Cancer Institute (Prof. Gerrit A. Meijer, head of the Division of Diagnostic Translational 
Oncology) to have access to fecal samples of individuals with similar characteristics of those 
planned to include in the miRFec study. More specifically, they have agreed to provide us a 
-80ºC frozen aliquot of left-over stool samples of up to 1245 individuals (both FIT positive 
and negative, obtained previously to colonoscopy, with complete colonoscopy and 
pathology characterization) recruited in the MOCCAS study18. Prevalence of colorectal 
neoplasms in such a cohort is very similar to the one observed in our study, so no potential 
bias is expected. The corresponding Material Transfer & Collaboration Agreement has been 
signed between both institutions.  
 
Following the completion of stage I of the study and based on the sensitivity and 
specificity results for both the miRFec score and FIT, we estimated the required sample size 
for stage II. This estimation assumes that the sensitivity of the miRFec score for detecting 
advanced colorectal neoplasms (i.e. CRC, advanced adenomas and advanced serrated 
lesions) will surpass that of FIT while maintaining equivalent specificity. The sample size 
calculation was conducted using the sensitivity of the miRFec score (36%) observed at the 
cut-off point corresponding to the same specificity (93%) achieved by FIT (at 20 µg Hb/ g of 
feces; sensitivity, 27%). To detect an absolute superiority margin of 6% with a one-sided 
test of proportions, an alpha risk of 0.025, and a beta risk of 0.10, we determined that a total 
of 2690 subjects will be required. 
 
12.2. Statistical analysis plan (SAP) 
Sensitivity [proportion of persons with disease who have a positive test] of the miRFec 
test will be compared with the corresponding figure obtained with FIT (cut-off level, 20 µg 
Hb/g of feces) for the detection of advanced colorectal neoplasm.  
Specificity [proportion of persons without disease who have a negative test] of the 
miRFec test will be compared with the corresponding figure obtained with FIT, with 
advanced colorectal neoplasms excluded and only non-advanced adenomas and negative 
results included (primary measure) and with only negative results included (secondary 
measure).  

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 22 of 40 
Discrimination capacity of the miRFec score and fecal hemoglobin concentration will 
be measured by the area under the receiver operating characteristic (ROC) curve for the 
detection of both advanced colorectal neoplasm and CRC. 
Other parameters to be calculated include positive predictive value [proportion of 
persons with disease among those with a positive test], negative predictive value [proportion 
of persons without disease among those with a negative test], and number needed to screen 
[number of persons who would need to be screened to identify one person with the disease] 
using either miRFec test or FIT.  
For test characteristics, 95% lower boundaries will be computed with the use of an 
exact binomial test. Lower 95% confidence limits for comparative analyses were computed 
with the use of a one-sided McNemar paired-comparisons test for the observed difference 
in sensitivity between the miRFec test and FIT. The Hanley-McNeil method will be used to 
calculate p values for the analysis of the ROC curve. 
No interim analysis of the data is planned.  
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 23 of 40 
13. ETHICS AND LEGAL ASPECTS 
The study will be carried out according to current legislation (14/2007 of July 3rd on 
Biomedical Research) and Helsinki Declaration guidelines approved by the 18th World 
Medical Association in Helsinki, Finland, in 1964, and amended in subsequent assemblies 
in Tokyo 1975, Venice 1983, Hong King 1989, Somerset West 1996, Edinburgh 2000, Seoul 
2008, and Fortaleza 2013. 
The current protocol has been elaborated according to the EU 2016/679 Regulation of 
European Parliament related to the protection of natural persons in terms of personal data 
treatment and free circulation of these data and ICH E6 (R2) Good Clinical Practice.  
The study has been submitted to the Research Ethics Committee (CEIm) of Hospital 
Clinic of Barcelona, and it will be submitted to the corresponding body of other participating 
centers if needed.  
All individuals will provide written informed consent to participate in the study (Annex 
2). 
With respect to the collaboration with the Netherlands Cancer Institute (Prof. Gerrit A. 
Meijer) (see Section 12.1. Sample size calculation), a Material Transfer & Collaboration 
Agreement has been signed between both institutions.  
 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 24 of 40 
14. DATA MANAGEMENT, CONFIDENTIALITY AND REGISTER FILE  
 
14.1. Confidentiality 
Treatment, communication and personal data transfer of all participants will comply 
with EU Regulation 2016/679 of the European Parliament and of the Council of April 27th, 
2016 on the protection of natural persons with regard to the processing of personal data and 
on the free movement of such data, and repealing Directive 95/46/EC (General Data 
Regulation), being mandatory since May 25th, 2018. The legal basis that justifies the 
processing of personal data is the consent given in this act, following the provisions of Article 
9 of EU Regulation 2016/679. 
Data will be collected and identified by a code, so no information to identify the 
participants will be included. Only study investigator and his/her collaborators with specific 
permission will be allowed to relate the data collected in the study with participant's medical 
record. 
Identity of participants will not be available to any other person except for a medical 
emergency or legal requirement. 
Health authorities, the Research Ethics Committee and authorized personnel by the 
study promoter may have access to identified personal information when necessary, to verify 
data and study procedures, but always maintaining confidentiality according to the current 
legislation. 
Only coded data will be transferred to third parties and other countries, which in no 
case will contain information that can directly identify the participant (such as name and 
surname, initials, address, social security number, etc.). If this transfer occurred, it would be 
only for the study and guaranteeing confidentiality. 
If a transfer of encrypted data is made outside the EU, either to entities related to the 
hospital center of participant, to service providers or researchers who collaborate with 
participant's physician, all data will be protected by safeguards such as contracts or other 
established mechanisms established by data protection authorities. 
Study promoter promises to process the data following EU Regulation 2016/679 and, 
therefore, to keep a record of treatment activities that are carried out and to perform a risk 
assessment treatment to know what measures should be applied and how to do it. 
In addition to the rights that the previous legislation already contemplated (access, 
modification, opposition and cancellation of data, deletion in the new Regulation), any 
participant can also limit the processing of incorrect data, request a copy or that the data 
provided for the study can be transferred to a third party (portability). To exercise these 
rights, participant should contact Principal Investigator of the study or the Data Protection 
Officer of the Hospital Clinic of Barcelona (protecciodades@clinic.cat). Moreover, participant 
has the right to contact the Data Protection Agency if he/she is not satisfied. 
Data already collected cannot be removed even if any participant leaves the study, to 
ensure the validity of the research and to come into effect with legal duties and drug 
authorization requirements. But no new data will be collected if he/she decides to stop 
participating. 
It is obligatory for researchers and study promoter to keep the data collected for the 
study at least 25 years after its completion. Subsequently, personal information will only be 
kept by the center for participant health care, and by the promoter for other scientific 
research purposes if the participant has given its consent to do so and if this is allowed by 
the law and applicable ethical requirements. 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 25 of 40 
14.2. Electronic case report form (e-CRF) 
All study data will be recorded in the e-CRF, which will be developed by Persei 
Vivarium using ReseaArch®, an electronic data capture solution that allows data collection 
in a simple way through forms that validate the information entered, ensuring its quality and 
reliability. It's built over a safe and trusted eHealth architecture with 21 CFR Part 11 
compliance. 
Data managed on this project will be anonymous. The forms will not collect data that 
allows a case identification.  
Main core features of ReseaArch® includes: 
▪ e-CRF. Allows the entry of cases. When the data is entered, the information is 
checked and validated. 
▪ Descriptive statistics. This module provides histograms, pie and bar charts, and 
statistics like average, deviation, etc. 
▪ 
Filters. Allows the analysis of subgroup of data and variables fulfilling specific or combined 
criteria.  
▪ 
Export data, in an excel file and manage the information by yourself.  
▪ 
Follow-up module. Includes two online reports: upcoming and overdue follow-ups.  
▪ 
Patients per hospital. Report that shows the number of patients included by each hospital.  
▪ 
Global view report. Online report showing the global project status.  
▪ 
Pending data. List of participants for whom the introduction of some information is still 
pending. 
Information recorded in the e-CRF will be structured in 4 blocks:  
▪ Enrollment, eligibility, demography, and kit delivery 
▪ Fecal sample collection 
▪ Study withdrawal, colorectal findings, and colonoscopy-related adverse events 
▪ Laboratory results 
Enrollment: country and participating center. Once this information recorded, a unique ID 
number will be automatically assigned to each patient. For anonymity reasons, name and 
surname, and medical record number of participants will be kept in a independent, local 
registry, not available for the coordinating center. 
Eligibility: includes fulfillment of inclusion and exclusion criteria.  
Demography: includes age, gender, ethnicity, number of first-degree relatives with CRC, 
number of first-degree relatives diagnosed with CRC before the age of 50, and indication for 
screening colonoscopy (i.e. average-risk or moderate-risk).  
Consent form and kit delivery: includes date of inclusion (which correspond to date of 
consent form signature) and code of fecal sample collecting kit (code bar of the kit will be 
scanned). 
Sample collection: date of fecal sample collection. 
Study withdrawal: if a participant is withdrawn from the study, the reason to do so will be 
recorded.  
Colorectal findings: includes number of diagnostic and therapeutic procedures performed, 
date of each procedure (either colonoscopy and/or surgery), cecal intubation, BBPS, 
number of polyps detected, number of polyps resected, number of invasive CRC detected, 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 26 of 40 
and number of invasive CRC resected. Pathology information from all detected lesions will 
also be recorded. 
Serious adverse events: related to screening colonoscopy (i.e. perforation, bleeding, or 
sedative-related adverse event) will be recorded. 
Laboratory: fecal hemoglobin concentration, fecal miR-421 expression, and fecal miR-27a-
3p expression. 
 
14.3. Data transfer 
Data introduced in the e-CRF must be consistent and according to the source 
documents (medical record, colonoscopy and pathology reports, among others). Only 
authorized personnel will introduce all data after receiving specific training on the use of e-
CRF. 
Any change in the initial recorded data will be justified and traceable so that the data 
is as clean and accurate as possible for analysis. Data quality is improved through a series 
of scheduled checks that automatically detect out-of-range or abnormal data. 
Data entry should not be delayed because of obtaining data source, trying to be as 
simultaneous as possible to avoid data loss. 
The e-CRF has been designed to require only the essential data for the study. 
 
14.4. Source documents 
A source document is understood as all observations or annotations recorded about 
clinical interventions, as well as all reports that are essential for the research study 
evaluation. Accordingly, source documents for this study include, but are not limited to, 
medical record, colonoscopy, pathology, and other medical reports. 
Whenever possible, the source document must be the original one; however, a clear, 
legible and exact copy can be accepted. All centers must keep these documents for auditoria 
or data monitoring. 
 
 
14.5. Responsibilities 
Only authorized and trained personnel will have access to the e-CRF.  
Data managers will guarantee access to the system for all authorized persons. 
The promoter is responsible for the consistency of data introduced in the e-CRF 
against the source document. 
The promoter is responsible for verification of information on the e-CRF and source 
document. 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 27 of 40 
15. BIOLOGICAL SAMPLES MANAGEMENT 
Participation in this study involves obtaining a stool sample.  
Participants agree that samples will be obtained according to biomedical research Law 
14/2007 and Royal Decree 1716/2011, which regulate the use of biological samples in 
research. 
Samples will be stored in the laboratory of Fundació Clínic per a la Recerca Biomèdica 
in Barcelona until they are used for this study. Once the study is finished, leftover samples 
will be destroyed unless participant expresses his/her consent (Annex 2) to store them at 
the Biobanc of Hospital Clínic-IDIBAPS at the registered collection number C. 0004006, and 
use them in future research.  
A code to identify fecal sample will be used. Only the study's Principal Investigator and 
his collaborators will be able to associate the sample with the participant. The information 
obtained from using these samples will be treated in the same way as the rest of the data 
obtained during this study. 
Biological samples transfer for this study is free and voluntary. That means that 
participant will have no rights to any possible commercial benefit from the findings that could 
result from biomedical research. 
Any relevant information that could affect participant health or his/her family members 
will be notified. If necessary, investigators will use the data on participant medical record to 
be in touch with him/her. However, participant willingness to receive no information will be 
respected.  
Additionally, the results obtained if genetic analyses are performed will not be 
communicated to participant or his/her physician, although he/she can request them by 
contacting the study's Principal Investigator. 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 28 of 40 
16. FUNDING 
 
Eiken Chemical Co., Ltd, Tokyo, donated the amount of 231,714 € in April 2021, as a 
sign of interest in this study. Subsequently, in March 2022, Eiken agreed to support it through 
a total funding of 455,769 € for stage I.   
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 29 of 40 
17. PUBLICATION POLICY 
The principal investigator is responsible for publishing the results of this study in 
medical journals and meetings. Local principal investigators as well as laboratory 
researchers involved in this project will be co-authors of any resulting publication and 
presentation. Centers with a large recruitment rate will be granted to add one additional 
investigator per each 1000 participants included in the study. Other investigators 
participating in the study will be part of a collective authorship, mentioned as "on behalf of 
the miRFec study investigators" and including his/her name in the corresponding annex.  
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 30 of 40 
18. REFERENCES 
  
1. 
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer 
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 
cancers in 185 countries. CA Cancer J Clin 2018;68:394-424. 
2. 
Young GP, Rabeneck L, Winawer SJ. The Global Paradigm Shift in Screening for 
Colorectal Cancer. Gastroenterology 2019;156:843-851. 
3. 
Steele RJ, Pox C, Kuipers EJ, Minoli G, Lambert R, International Agency for 
Research on C. European guidelines for quality assurance in colorectal cancer 
screening and diagnosis. First Edition--Management of lesions detected in colorectal 
cancer screening. Endoscopy 2012;44 Suppl 3:SE140-50. 
4. 
Bibbins-Domingo K. Colorectal Cancer Screening Recommendations-Reply. JAMA 
2016;316:1717. 
5. 
Gupta S, Halm EA, Rockey DC, et al. Comparative effectiveness of fecal 
immunochemical test outreach, colonoscopy outreach, and usual care for boosting 
colorectal cancer screening among the underserved: a randomized clinical trial. 
JAMA Intern Med 2013;173:1725-32. 
6. 
Robertson DJ, Lee JK, Boland CR, et al. Recommendations on Fecal 
Immunochemical Testing to Screen for Colorectal Neoplasia: A Consensus 
Statement by the US Multi-Society Task Force on Colorectal Cancer. 
Gastroenterology 2017;152:1217-1237. 
7. 
Segnan N, Patnick J, von Karsa L. European guidelines for quality assurance in 
colorectal cancer screening and diagnosis. Luxembourg: Publications Office of the 
European Union, 2010. 
8. 
Quintero E, Castells A, Bujanda L, et al. Colonoscopy versus fecal immunochemical 
testing in colorectal-cancer screening. N Engl J Med 2012;366:697-706. *Both Dr. 
Quintero and Dr. Castells share first authorship. 
9. 
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 
2004;116:281-97. 
10. 
Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Nat Rev 
Cancer 2006;6:259-69. 
11. 
Di Leva G, Croce CM. The Role of microRNAs in the Tumorigenesis of Ovarian 
Cancer. Front Oncol 2013;3:153. 
12. 
Link A, Balaguer F, Shen Y, et al. Fecal MicroRNAs as novel biomarkers for colon 
cancer screening. Cancer Epidemiol Biomarkers Prev 2010;19:1766-74. 
13. 
Duran-Sanchon S, Moreno L, Auge JM, et al. Identification and Validation of 
MicroRNA Profiles in Fecal Samples for Detection of Colorectal Cancer. 
Gastroenterology 2020;158:947-957. 
14. 
Duran-Sanchon S, Moreno L, Gomez-Matas J, et al. Fecal MicroRNA-Based 
Algorithm Increases Effectiveness of Fecal Immunochemical Test-Based Screening 
for Colorectal Cancer. Clin Gastroenterol Hepatol 2021;19:323-330. 
15. 
Kaminski MF, Thomas-Gibson S, Bugajski M, et al. Performance measures for lower 
gastrointestinal endoscopy: a European Society of Gastrointestinal Endoscopy 
(ESGE) Quality Improvement Initiative. Endoscopy 2017;49:378-397. 
16. 
Lai EJ, Calderwood AH, Doros G, Fix OK, Jacobson BC. The Boston bowel 
preparation scale: a valid and reliable instrument for colonoscopy-oriented research. 
Gastrointest Endosc 2009;69:620-5. 
17. 
Loughrey MB, Ings G, Dickey W, Owen TA, Coleman HG. Evaluating the impact of 
2020 post-polypectomy surveillance guidelines in the Northern Ireland bowel cancer 
screening programme. Gut 2021;70:226-228. 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 31 of 40 
18. 
van Lanschot MC, Carvalho B, Coupe VM, van Engeland M, Dekker E, Meijer GA. 
Molecular stool testing as an alternative for surveillance colonoscopy: a cross-
sectional cohort study. BMC Cancer 2017;17:116. 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 32 of 40 
ANNEX 1. Information sheet 
 
PATIENT INFORMATION SHEET 
 
STUDY TITLE: Prospective, multicenter, comparative, paired study to validate a microRNA- based 
fecal test for colorectal cancer screening, surveillance and diagnosis. The miRFec study. 
 
SPONSOR CODE: miRFec001 
 
SPONSOR: Fundació Clínic per a la Recerca Biomèdica (Clinic Foundation for Biomedical Research) 
 
PRINCIPAL INVESTIGATOR: Antoni Castells, MD, PhD; Gastroenterology Department, Hospital Clinic 
of Barcelona. Phone number: + 34 93 227 57 03  
 
CENTRE: Hospital Clinic of Barcelona 
 
INTRODUCTION 
We invite you to participate in a research study. The study has been approved by the Research Ethics 
Committee according to the current legislation (Ley de Investigación Biomédica 14/2007). 
Before you decide whether or not you wish to participate in this study, it is important for you to 
understand why the research is being done and what it will involve. 
Please take your time to read the following information carefully and ask us if there is anything that 
is not clear or if you would like more information. You can also discuss it with friends and relatives 
if you wish.   
If you decide to take part of the study, you will be given a copy of this information sheet and your 
signed consent form. 
 
PARTICIPATION 
Participation in this study is voluntary and you can decide whether or not you want to participate in 
it. Whatever your decision, it will not affect your relationship with the staff caring. If you wish to 
withdraw from the study once it has started, you can do so at any time without having to give a 
reason. 
 
GENERAL DESCRIPTION OF THE STUDY 
Colorectal cancer is the third most common cancer and the second leading cause of cancer-related 
death in the world. Although 90% of patients diagnosed at early stages have an overall survival of 
more than 5 years, this figure decreases to 10% in patients diagnosed at advanced stages with 
distant metastasis. In that sense, evidence from several studies has shown that CRC screening is 
effective and cost-effective in general population.  
The most extended strategy worldwide in population-based organized colorectal cancer screening 
programs is fecal immunochemical testing (FIT), which looks for occult blood in the stool, an early 
sign of cancer. While exams such as colonoscopy, another exam used for colorectal cancer 
screening, detect both cancer and premalignant lesions –so-called adenomatous polyps or 
adenomas–, FIT mainly identify cancer because of their low sensitivity for the detection of advanced 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 33 of 40 
adenomas. Moreover, the relatively low specificity results in a high false-positive rate, leading to a 
significant number of unnecessary colonoscopies.  
In the last years, the use of FIT has been proposed for other indications, besides CRC screening. 
Indeed, colonoscopy surveillance is being substituted by FIT in those patients in whom non-
advanced colorectal lesions, and it has been suggested a similar surveillance strategy for those 
patients with advanced colorectal lesions. Finally, FIT is also commonly used to triage those 
individuals with colorectal symptoms who may benefit from a diagnostic colonoscopy.  
A different approach to optimize CRC screening, colorectal surveillance and colorectal diagnosis is 
the use of biomarkers other than hemoglobin, such as microRNAs, which can be detected in either 
blood or feces and they may contribute to overcome the above-mentioned limitations. 
We aim to validate the results recently obtained by our research group, which demonstrated that 
two microRNAs (miR-421 and miR-27a-3p) along with fecal hemoglobin concentration –the miRFec 
test– identify patients with advanced colorectal neoplasia (that is, cancer or advanced polyps) more 
accurately than hemoglobin concentration along among FIT-positive individuals. 
The main goal of present study is to compare the sensitivity of the miRFec test with respect to FIT 
for the detection of patients with advanced colorectal neoplasia among individuals participating in 
colorectal cancer screening, colorectal surveillance and colorectal diagnosis. We will also compare 
specificity, detection rate and cost-effectiveness of the miRFec test with respect to FIT, and 
sensitivity for detection of cancer and advanced polyps separately. 
If you accept to participate in the study, our staff will provide you with a fecal sample collection kit 
along with instructions for sample collection. The sample must be collected at home before starting 
bowel preparation, more specifically between 24-72 hours before the day that colonoscopy is 
scheduled.  
The sample must be collected before start taking the laxatives prior to the colonoscopy you should 
undergo. Once the sample is collected, you must keep it on the fridge until you come to the hospital 
to perform the colonoscopy. The endoscopy staff will be in charge of collecting and storing the 
sample. 
The study population will consist of individuals aged 50 to 75 without family history of colorectal 
cancer and individuals aged 18 or older with such a familial background, all of them referred to 
colonoscopy-based colorectal cancer screening, as well as those individuals older than 18 with 
personal history of colorectal cancer and colorectal polyps referred to colonoscopy surveillance and 
those with colorectal symptoms referred to diagnostic colonoscopy.  
Eligible individuals will undergo two different examinations –FIT and miRFec test– in a unique stool 
sample collected at home, which results will be compared to those obtained from the colonoscopy 
you should undergo.  
The study will be carried out in two consecutive stages: stage I to calculate the most adequate 
threshold of the miRFec test, in which 2700 individuals will be included, and stage II to verify this 
cut-off value in 2690 additional individuals. 
 
BENEFITS AND RISKS DERIVED FROM YOUR PARTICIPATION IN THE STUDY 
There is no direct benefit for individuals participating in the study. However, results obtained in this 
study will allow us to improve diagnostic capacity of current screening methods, and they may 
benefit individuals like you in the near future.  

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 34 of 40 
Participation in this study will not represent any increased risk in addition to complications related 
to the colonoscopy you should undergo (use of laxative for bowel preparation, adverse reaction to 
sedation, or bleeding and perforation if any lesion should be resected). 
Participation in the study will not represent any additional medical visit or examination. 
Analyses carried out in the stool sample you provide us will be done in our laboratory, so there is 
no risk for pregnant or lactating women.  
Your responsibilities will only consist of collecting the stool sample at home, keep it on the fridge, 
and bring it to the hospital the day your colonoscopy is performed. 
 
ALTERNATIVE TREATMENTS 
Your participation in the study does not change the treatment prescribed by your physician, since 
you should always take laxatives as bowel preparation for colonoscopy. 
 
CONFIDENTIALITY AGREEMENT 
The Fundació Clínic per a la Recerca Biomèdica (Clinic Foundation for Biomedical Research), with CIF 
G-59319681, as responsible for your data processing, informs you that the treatment, 
communication and transferring of personal data of all participants will comply with EU Regulation 
2016/679 of the European Parliament and with April 27, 2016 Council, regarding the protection of 
people concerning the processing and free circulation of personal data, being compulsory since May 
25, 2018. The legal basis that justifies the processing of your data is the consent that appears in this 
act, according to article 9 of EU Regulation 2016/679. 
Data for these studies will be collected and identified by a code, so no information to identify the 
participants will be included. Only study's Principal Investigator and his collaborators with specific 
permission will be allowed to relate the data collected in the study with your medical record. 
Your identity will not be available to any other person except for a medical emergency or legal 
requirement. The health authorities, the Research Ethics Committee and authorized personnel by 
the study Promoter may have access to your identified personal information when necessary, to 
verify data and study procedures, but always maintaining confidentiality according to the current 
legislation. 
Only coded data will be transferred to third parties and other countries, which in no case will contain 
information that can directly identify the participant (such as name and surname, initials, address, 
social security number, etc.). If this transfer occurs, it would be only for the study and guaranteeing 
confidentiality. 
If a transfer of encrypted data is made outside the EU, either to entities related to the hospital center 
where you participate, to service providers or researchers who collaborate with your doctor, your 
data will be protected by safeguards such as contracts or other established mechanisms established 
by data protection authorities. 
In addition to the rights that the previous legislation already contemplated (access, modification, 
opposition and cancellation of data, deletion in the new Regulation) you can now also limit the 
processing of incorrect data, request a copy or that the data provided for the study can be 
transferred to a third party (portability). To exercise these rights, or if you want to know more about 
confidentiality, you should contact the Principal Investigator of the study or the Data Protection 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 35 of 40 
Officer of the Fundació Clínic per a la Recerca Biomèdica through protecciodades@clinic.cat. They 
also have the right to contact the Data Protection Agency if you are not satisfied. 
Data already collected cannot be removed even if you leave the study, to ensure the validity of the 
research and to come into effect with legal duties and drug authorization requirements. But no new 
data will be collected if you decide to stop participating. 
It is mandatory for the Investigator and the Promoter to keep the data collected for the study at 
least 25 years after its completion. Subsequently, personal information will only be kept by the 
center for your health care and by the Promoter for other scientific research purposes if the patient 
has given its consent to do so, and if this is allowed by the law and applicable ethical requirements. 
 
ECONOMIC COMPENSATION 
Your participation in the study will not represent any economic cost to you. You will not have to pay 
for the study tests. On the other hand, you will not receive any economic compensation for 
participating in the study. 
 
COLLECTION AND USE OF BIOLOGICAL SAMPLES  
Participation in this study involves obtaining a stool sample.  
By signing this document, you agree that the samples will be obtained from the present study, 
according to biomedical research Law 14/2007 and Royal Decree 1716/2011, which regulates the 
use of biological samples in research. 
Samples will be stored in the laboratory of Fundació Clínic per a la Recerca Biomèdica in Barcelona 
until they are used for this study. Once the study is finished, leftover samples will be destroyed 
unless you express your consent to store them at the Biobanc of Hospital Clínic-IDIBAPS at the 
registered collection number C. 0004006, and use them in future research.  
A code to identify your sample will be used. Only the study's Principal Investigator and his 
collaborators will be able to associate the sample with you. The information obtained from using 
these samples will be treated in the same way as the rest of the data obtained during this study. 
Biological samples transfer for this study is free and voluntary. That means that you will have no 
rights to any possible commercial benefit from the findings that could result from biomedical 
research. 
Any relevant information that could affect your health or your family members will be notified. If 
necessary, we will use the data on your medical record to be in touch with you. However, your 
willingness to receive no information will be respected. If you wish, please check the box found on 
the consent form.  
Additionally, the results obtained if genetic analyses are performed will not be communicated to 
you or your physician, although you can request them by contacting the study's Principal 
Investigator. 
 
OTHER RELEVANT INFORMATION 
Any new information regarding the test used in the study that may affect your willingness to 
participate, discovered during your participation, will be communicated to you as soon as possible. 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 36 of 40 
 
 
If you decide to withdraw your consent, no new data will be added to the database and you can 
demand the destruction of all identifiable samples previously retained to avoid further analysis. 
You should also know that you can be excluded from the study if the Promoter or study Investigators 
consider it appropriate either for safety reasons, for any adverse event or because they consider 
that you are not complying with the established procedures. Anyway, you will receive an adequate 
explanation of the reason for your withdrawal from the study. 
 By signing the attached consent form, you accept to comply with the study procedures that have 
been set forth to you. 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 37 of 40 
ANNEX 2. Participant consent form 
 
PATIENT CONSENT FORM 
 
Study title: Prospective, multicenter, comparative, paired study to validate a microRNA- based 
fecal test for colorectal cancer screening, surveillance and diagnosis. The miRFec study.  
 
Protocol code: miRFec001 (version 5; February 5th, 2025) 
 
Me, (name and surname) ……………………………………………………………………………………………........... 
 
- I have read the information sheet about the study. 
- I have had the opportunity to ask questions about the study. 
- I have received enough information about the study. 
- I have talked to (investigator's name) ………………………………………………………………………………........ 
- I comprehend that my participation is voluntary.  
- I comprehend that I can withdraw from the study at any time:  
 
- Whenever 
 
- Without giving any reason 
 
- Without my medical care or legal rights being affected. 
 
- According to the provisions of EU Regulation 2016/679 of the European Parliament and of the 
April 26th, 2016 Council about the protection of natural persons regarding personal data 
processing and free circulation of data, I declare that I have been informed of the existence of a 
file or a personal data processing, the purpose of collecting these data and the information 
receivers. 
 
- I give my consent to participate in this study. 
 
 
 
 
Participant signature  
 
 
 
Investigator signature 
Date: ___/___/_____  
 
 
 
Date: ___/___/_____ 
 
Wish to be informed of any relevant information obtained from the study that might be important 
for my health: 
 
 
 
 
☐ YES          ☐ NO        
 
I give my consent to store and keep remaining samples, so they can be used in future research: 
 
 
 
 
☐ YES         ☐ NO         
 
 
                       
  
Participant signature  
 
 
 
Investigator signature 
Date: ___/___/_____  
 
 
 
Date: ___/___/_____ 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 38 of 40 
 
ANNEX 3. Instructions for fecal sample collection 
 
 
 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 39 of 40 
ANNEX 4. Staging according to American Joint Committee on Cancer (8th version)17 
 
Primary tumor (T) 
▪ Tx: primary tumor cannot be assessed 
▪ T0: no evidence of primary tumor 
▪ Tis: carcinoma in situ, intramucosal carcinoma (involvement of lamina propria with no 
extension through muscularis mucosae) 
▪ T1: tumor invades submucosa (through the muscularis mucosae but not into the 
muscularis propria) 
▪ T2: tumor invades muscularis propria 
▪ T3: tumor invades through the muscularis propria into the pericolorectal tissues 
▪ T4a: tumor invades through the visceral peritoneum (including gross perforation of the 
bowel through tumor and continuous invasion of tumor through areas of inflammation to 
the surface of the visceral peritoneum) 
▪ T4b: tumor directly invades or adheres to other adjacent organs or structures 
 
Regional lymph nodes (N) 
▪ Nx: regional lymph nodes cannot be assessed 
▪ N0: no regional lymph node metastasis 
▪ N1: metastasis in 1-3 regional lymph nodes 
▪ N1a: metastasis in 1 regional lymph node 
▪ N1b: metastasis in 2-3 regional lymph nodes 
▪ N1c: no regional lymph nodes are positive but there are tumor deposits in the 
subserosa, mesentery or nonperitonealized pericolic or perirectal/mesorectal tissues 
▪ 
N2: metastasis in 4 or more regional lymph nodes: 
▪ N2a: metastasis in 4-6 regional lymph nodes 
▪ N2b: metastasis in 7 or more regional lymph nodes 
 
Distant metastasis (M) 
▪ M0: no evidence of tumor in other sites or organs 
▪ M1: distant metastasis: 
▪ M1a: metastasis confined to 1 organ or site without peritoneal metastasis 
▪ M1b: metastasis to 2 or more sites or organs is identified without peritoneal 
metastasis 
▪ M1c: metastasis to the peritoneal surface is identified alone or with other site or organ 
metastasis 

 
 
Study protocol 
The miRFec study. Version 5. Date: February 5th, 2025 
Page 40 of 40 
 
Stage I 
T1 
N0 
M0 
T2 
N0 
M0 
Stage IIa 
T3 
N0 
M0 
Stage IIb 
T4a 
N0 
M0 
Stage IIc 
T4b 
N0 
M0 
Stage IIIa 
T1 
N1 
M0 
T1 
N1c 
M0 
T1 
N2a 
M0 
T2 
N1 
M0 
T2 
N1c 
M0 
Stage IIIb 
T1 
N2 
M0 
T2 
N2a 
M0 
T2 
N2b 
M0 
T3 
N1 
M0 
T3 
N1c 
M0 
T3 
N2a 
M0 
T4 
N1 
M0 
T4 
N1c 
M0 
Stage IIIc 
T3 
N2b 
M0 
T4a 
N2a 
M0 
T4a  
N2b 
M0 
T4b 
N1 
M0 
T4b 
N2 
M0 
Stage IV 
Tx 
Nx 
M1