The Role of Gut Microbiome and Chronic Inflammation in Young-onset Colorectal Cancer: Next-generation Sequencing (NGS) as a Screening Method
Conditions: Colorectal Cancer
Sponsor: Fakultas Kedokteran Universitas Indonesia

RESEARCH PROPOSAL 
 
 
 
 
 
THE ROLE OF GUT MICROBIOME AND CHRONIC INFLAMMATION IN 
YOUNG-ONSET COLORECTAL CANCER 
Next-Generation Sequencing (NGS) as screening method 
 
 
 
 
RESEARCH TEAM 
MURDANI ABDULLAH 
SAFARINA G MALIK 
ARI FAHRIAL SYAM 
YUSRA 
FAUZI YUSUF 
 
 
 
 
 
 
FACULTY OF MEDICINE 
UNIVERSITAS INDONESIA 
 
24 April 2019 

2 
 
INTRODUCTION 
 
Background 
In 2025 the World Health Organization (WHO) estimates that cancer will have a higher mortality 
rate than coronary heart disease and stroke. It is estimated there are more than 20 million new cases 
of cancer each year in low- and middle-income countries due to global epidemiological changes. 
Colorectal cancer is the third most prevalent cancer worldwide and is in the fourth place in cancer 
mortality rate. The incidence of colorectal cancer is estimated to be 1.2 million each year with 
more than 630.000 number of deaths, accounting for 8% of deaths caused by cancer. In 2030 it is 
estimated there will be 2.2 million number of new cases and 1.1 million number of deaths caused 
by colorectal cancer. Based on Jakarta Cancer Registry in 2012 in Indonesia, colorectal cancer is 
the second most common cancer in men and the fourth most common in women. The incidence of 
colorectal cancer was 3.15 per 100.000 of populations in women and 4.13 per 100.000 of 
populations in men. 
 
Previous studies indicated that colorectal cancer patients in Indonesia were younger compared to 
developed countries and were sporadic. Sporadic colorectal cancer generally resulted from somatic 
mutation without any correlation to family history. In comparison to patients in developed 
countries, colorectal cancer patients in Indonesia also showed differences in several characteristics 
including distal localization (rectum), higher clinical staging, and poor prognosis. 
 
Increasing number of colorectal cancer cases make a profound impact on national health 
expenditures. In the era of Jaminan Kesehatan Nasional / JKN (the National Health Insurance in 
Indonesia), there are limitations on budgets for colorectal cancer management. Thus, numerous 
efforts are needed on the early screening and prevention of colorectal cancer.  
 
Gastrointestinal system comprises of more than 200 m2 of mucosal surface. The immune system 
in gastrointestinal mucosa has been studied extensively until this day. The microflora or the 
microorganism in gastrointestinal system, also commonly known as the gut microbiome, is a part 
of the immunity system in gastrointestinal mucosa. Around 20% of malignancy are linked to 
microorganism. The gut microbiome is suspected to have a role in genetic and epigenetic changes 
that cause dysplasia, clonal expansion, tumor growth, and cancer. In addition, chronic 

3 
 
inflammation is also suspected to have a role in colorectal carcinogenesis. One of the chronic 
inflammation markers linked with carcinogenesis is NF-ҡB. 
 
Several studies showed that dysbiosis or imbalance of gut microbiome is linked with mucosal 
barrier damage, chronic inflammation and production of carcinogenic metabolite causing 
neoplasm. Thus far, the association between gut microbiome and colorectal cancer has provided 
an opportunity to develop new strategies in the screening method of colorectal cancer. 
 
Non-invasive methods for colorectal cancer screening are currently being developed to replace 
colonoscopy. One of the screening methods is fecal immunochemical test (FIT) using fecal 
samples. This method has a sensitivity of 87.5% in detecting colorectal cancer cases. Another 
screening method uses blood sample to evaluate carcinoembryonic antigen (CEA) level in blood 
serum. An increase in CEA serum level of more than 5 ng/mL is found in 17-47% of colorectal 
patients and is associated with poor prognosis.  
 
Research Objectives 
General Objectives 
To investigate the role of gut microbiome pattern and inflammation marker NF-ҡB in colorectal 
cancer patients 
 
Specific Objectives 
1. To obtain data on gut microbiome pattern on young-onset colorectal cancer patients and 
colorectal cancer patients aged >50 years old. 
2. To explore the role of inflammation marker NF-ҡB on young-onset colorectal cancer 
patients and colorectal cancer patients aged >50 years old. 
3. To obtain CEA values of young-onset colorectal cancer patients and colorectal cancer 
patients aged >50 years old. 
4. To obtain data on gut microbiome which could be used as screening methods in patients 
suspected with colorectal cancer  
 
 

4 
 
Hypothesis 
1. There are differences in gut microbiome pattern on young-onset colorectal cancer patients 
and colorectal cancer patients aged >50 years old compared to healthy individuals 
2. There are differences in NF-ҡB on young-onset colorectal cancer patients and colorectal 
cancer patients aged >50 years old compared to healthy individuals 
3. There are differences in CEA values on young-onset colorectal cancer patients and 
colorectal cancer patients aged >50 years old compared to healthy individuals 
 
Research Benefits 
For Researchers 
Researchers will expand their knowledge on gut microbiome pattern in colorectal cancer 
patients. 
 
For Institutions 
This study will provide a contribution of science in national and international scale. The result 
of this study could be used as a guideline on utilizing gut microbiome data on colorectal cancer 
management.  
 
For Communities 
The result of this study is expected to improve the management of colorectal cancer as a 
screening method that would be applicable in daily clinical practice. 
 
 
 

5 
 
METHODOLOGY 
 
 
Study Design 
This is a cross-sectional study. 
 
Time and Place 
This study is conducted in Dr. Cipto Mangunkusumo Hospital: Digestive Endoscopy Center, 
Department of Internal Medicine, Department of Clinical Pathology, Department of Anatomical 
Pathology, and Eijkman Institute of Molecular Biology from May 2019 to May 2022. 
 
Population 
Target Population 
Patient suspected with colorectal cancer in Indonesia. 
Accessible Population 
Patients suspected with colorectal cancer who come to Digestive Endoscopy Center in Dr. 
Cipto Mangunkusumo Hospital to conduct colonoscopy procedure and histopathological 
examination. 
 
Subject Criteria 
Inclusion Criteria 
1. Age ≥ 35 years old 
2. Suspected with colorectal cancer and undergoing a colonoscopy procedure 
3. No history of colorectal cancer treatment 
 
Exclusion Criteria 
1. Unwilling to provide fecal and blood sample 
2. Incomplete colonoscopy procedure due to any reasons 
 
Estimated Sample Size 
Since this is a pilot study to obtain gut microbiome pattern on colorectal cancer patients, the 
number of samples is determined by the researchers: 100 subjects for neoplasm and 50 subjects 
for non-neoplasm (according to histopathology report). 

6 
 
Randomisation  
The sampling will not be randomized. All patients suspected with colorectal cancer who come to 
Digestive Endoscopy Center in Dr. Cipto Mangunkusumo Hospital to conduct colonoscopy 
procedure and histopathological examination who fulfilled the criteria will be recruited for this 
study and will undergo a series of examinations 
 
Operational Definition 
 
No.  
Variable  
Definition  
Source  
Scale  
1.  
Patient 
suspected with 
colorectal 
cancer  
Patient suspected with colorectal 
cancer by clinician based on 
clinical findings such as 
gastrointestinal bleeding, chronic 
constipation, chronic diarrhea, 
nonspecific abdominal pain, 
weight loss, palpable abdominal 
mass 
Clinical 
judgement 
Nominal 
2. 
Age 
Patient’s age (in years) 
 
Medical record 
3. 
Sex 
Patient’s sex 
Medical record 
Nominal 
4. 
Family history 
of colorectal 
cancer in a 
first-degree 
relative  
First-degree relative: parents, 
sibling, children 
Medical record 
and history 
taking 
Nominal 
1= No 
2= Yes 
5. 
History of 
smoking 
Past or current history of smoking  Medical record 
and history 
taking 
Nominal 
1= No 
2= Yes 
4. 
FIT results 
The result of Fecal 
immunochemical test (FIT) 
Fecal immuno-
chemical test 
(FIT) 
Nominal 
1= Negative 
2= Positive 
5. 
Gut 
microbiome 
results 
The result of gut microbiome 
examination 
Next Generation 
Sequencing 
(NGS) 
Nominal 
1: Fusobacterium 
2: Bacteroides 
3. Lachnospiraceae 

7 
 
6. 
NF-ҡB results The results of NF-ҡB 
examination: positive if 
accumulated score ≥3 
Examination 
with immune-
histochemical 
method 
Nominal 
1= Negative 
2= Positive 
7.        CEA results 
The results of Carcinoembryonic 
antigen (CEA) examination 
Examination 
with ELISA 
method 
Nominal 
1= Negative 
2= Positive 
7 
Histo-
pathological 
results   
The results of histopathological 
examination from biopsy 
samples are used as gold 
standard of colorectal cancer 
diagnosis  
Medical record  Nominal  
1 = Colorectal cancer 
2 = Non-colorectal 
cancer 
 
 
Ethical Approval 
This study will be obtained ethical approval from the Ethics Committee of Faculty of Medicine, 
University of Indonesia. 
 
Research Flow 
Patients suspected with colorectal cancer who come to Digestive Endoscopy Center in Dr. Cipto 
Mangunkusumo Hospital to conduct colonoscopy procedure and histopathological examination 
who fulfilled the criteria will be recruited for this study and will undergo a series of examinations 
Each patient will be given an explanation of this study and will be asked his/her willingness to 
participate on this study by signing the informed consent form. Baseline characteristics data will 
be obtained from medical record. Before the colonoscopy procedure is conducted, each patient 
will undergo a score assessment, blood sampling, and fecal sampling.  
1. Score Assessment 
Asia Pacific Colorectal Screening (APCS) is a validated tool to predict the risk of colorectal 
cancer in asymptomatic Asian population. The scoring system comprises of three 
categories: low risk (score 0-1), moderate risk (score 2-3) and high risk (score 4-7). Patients 
with moderate and high risk will undergo further examinations 
 
 
 

8 
 
Table. Asia Pacific Colorectal Screening (APCS).  
Risk Factor 
Criteria 
Score 
Age (in years) 
< 50 
0 
50 – 69  
2 
≥ 70  
3 
Sex 
Female 
0 
Male 
1 
Family history of colorectal 
cancer in a first-degree 
relative 
Absent 
0 
Present 
2 
History of smoking 
Never 
0 
Current or past 
1 
 
2. Blood Sampling 
Blood samples will be taken before colonoscopy procedure to evaluate the level of serum 
CEA by ELISA method and to evaluate the presence of NF-ҡB by immunohistochemical 
method. 
a. Carcinoembryonic antigen (CEA) is a well-known marker for colorectal cancer. A 
pre-treatment serum CEA level of ≥ 5 ng/mL is associated with poor prognosis in 
colorectal cancer patients. 
b. NF-ҡB is a chronic inflammation marker found in colorectal cancer patients 
3. Fecal Sampling 
Fecal samples will be taken before colonoscopy procedure to be tested for FIT and to 
evaluate the gut microbiome. 
a. Fecal immunochemical test (FIT) is a recommended screening method for colorectal 
cancer. Detection of hemoglobin over a certain level in fecal samples indicated a 
positive FIT. Patients with positive FIT will undergo further examinations. 
b. Gut microbiome examination will be conducted with next generation sequencing 
(NGS) method. 
 
 

9 
 
 
Blood samples and fecal samples will be tested in Department of Clinical Pathology Dr. Cipto 
Mangunkusumo Hospital, while gut microbiome will be evaluated in Eijkman Institute of 
Molecular Biology. Colonoscopy procedure will be conducted in Digestive Endoscopy Center Dr. 
Cipto Mangunkusumo Hospital. Histopathological examination be conducted in Department of 
Anatomical Pathology Dr. Cipto Mangunkusumo Hospital. The results of colonoscopy procedures 
and histopathology report will be obtained from medical record. 

10 
 
Gut microbiome examination from fecal samples will be initiated by DNA isolation. After 
extraction material is obtained, PCR will be performed with 16S RNA Sequencing. Metagenomic 
study will be carried out by analysing prokaryotic 16S ribosomal RNA gene (16S rRNA) which 
has a length of 1500 base pairs and consists of 9 variable regions that are between conserved 
regions. Variable regions that are generally used for phylogenetic analysis are V3 and V4 for 
classification to the level of genus or species. To obtain gut microbiome pattern, Next-Generation 
Sequencing (NGS) examination will be conducted. 
 
 
 
 

11 
 
TIMELINE 
 
First Year 
No 
Activities 
Month 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
1 
Obtaining ethical 
approval 
X 
X 
 
 
 
 
 
 
 
 
 
 
2 
Obtaining location 
permit 
 
X 
X 
 
 
 
 
 
 
 
 
 
3 
Sampling 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
4 
FIT, CEA, NF-kB, 
gut microbiome 
examinations 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Second Year 
No 
Activities 
Month 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
1 
Sampling 
X 
X 
X 
 
 
 
 
 
 
 
 
 
2 
FIT, CEA, NF-kB, 
gut microbiome 
examinations 
X 
X 
X 
 
 
 
 
 
 
 
 
 
3 
Data analysis and 
report writing 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Third Year 
No 
Activities 
Month 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
1 
Data analysis and 
report writing 
X 
X 
X 
 
 
 
 
 
 
 
 
 
2 
Scientific publication 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 

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