Biomarker Discovery in Lung Cancer - Malaysia
Conditions: Lung Cancer
Sponsor: University of Malaya

Biomarker Discovery in Lung Cancer – Malaysia 
CA-LUC-MY-B2 Protocol Version 2.0 20240116 
 
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 Document Number 
Version 
Effective Date (YYYY-MM-DD) 
PRO-MAS-002 
2.0 
2024-01-16 
 
Protocol Title: 
Biomarker Discovery in Lung Cancer-Malaysia 
 
Protocol Version: 
Version 2.0 
 
Protocol Date (YYYY-MM-DD): 
2024-01-16 
 
 
Industry-sponsored research with grant given by : 
MiRXES Lab Pte Ltd 
11 Biopolis Way, 
Singapore 138667 
 
VERSION HISTORY 
No. 
Description & Reason of Changes 
Version 
Effective 
Date 
1 
Initial Version 
1.0 
31 Mar 2023 
2 
Updated investigator-initiated research to industry-sponsored 
research. 
Removal of pneumonia as part of the inclusion criteria. 
Blood sample requirements updated. 
AE/SAE definitions and reporting 
Updated list of study team members. 
Clarification on Visit 2 blood collection visit.  
Administrative changes to Inclusion/Exclusion Criteria  
Removal of Appendix B,C,D 
2.0 
16 Jan 2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Title: Biomarker Discovery in Lung Cancer-Malaysia 
 
Protocol Number: PRO-MAS-002 
 
Protocol Version/ Date: Version 2.0/ 16-Jan-2024 
 
Sponsor Name: MiRXES Lab Pte. Ltd. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
1 
LIST OF ABBREVIATIONS ............................................................................................................ 5 
2 
BACKGROUND INFORMATION AND STUDY RATIONALE ........................................................ 6 
3 
STUDY OBJECTIVES AND OUTCOME ........................................................................................ 8 
3.1 
Study Objectives ..................................................................................................................... 8 
3.2 
Study Hypotheses ................................................................................................................... 8 
3.3 
Study Outcome ....................................................................................................................... 8 
4 
STUDY DESIGN ............................................................................................................................ 8 
5 
STUDY POPULATION ................................................................................................................. 10 
5.1 
Criteria for Recruitment ......................................................................................................... 10 
5.1.1 
Cohort A ........................................................................................................................ 10 
5.1.2 
Cohort B ........................................................................................................................ 10 
5.2 
Inclusion Criteria ................................................................................................................... 11 
5.2.1 
Inclusion Criteria for Each Cohort ................................................................................. 11 
5.3 
Exclusion Criteria .................................................................................................................. 11 
5.4 
Subject Withdrawal ............................................................................................................... 11 
5.5 
Blood Sample Requirements ................................................................................................ 12 
5.6 
Possible Risks and Discomfort to Subjects .......................................................................... 12 
5.7 
Adverse Events ..................................................................................................................... 12 
5.8 
Possible Benefits .................................................................................................................. 13 
5.9 
Costs and Payments if Participating in the Study ................................................................. 13 
6 
STUDY PROCEDURES AND ASSESSMENT ............................................................................. 13 
6.1 
Enrolment ............................................................................................................................. 13 
6.2 
Data Collection ..................................................................................................................... 13 
6.3 
Blood Samples Collection ..................................................................................................... 14 
6.4 
Clinical Specimens and Key Data Requirements ................................................................. 14 
6.5 
Biomolecules Extraction and Sequencing ............................................................................ 14 
6.5.1 
miRNA .......................................................................................................................... 15 
6.5.2 
Cell-free DNA (cfDNA) .................................................................................................. 15 
6.5.3 
Others ........................................................................................................................... 15 
7 
ANALYTICAL METHODS ............................................................................................................ 15 
8 
DATA HANDLING AND RECORD KEEPING .............................................................................. 15 
8.1 
Clinical Data Collection Forms .............................................................................................. 15 
8.2 
Data Entry and Storage ........................................................................................................ 16 
9 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ............................................................... 16 
10 
ETHICAL CONSIDERATIONS ..................................................................................................... 16 
10.1 
Institutional Review Board (IRB) ........................................................................................... 16 
10.2 
MiRXES’ IRB ........................................................................................................................ 16 
10.3 
Ethical Conduct of the Trial ................................................................................................... 16 

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10.4 
Confidentiality of Data and Patient Records ......................................................................... 17 
10.5 
Addressing Vulnerability ....................................................................................................... 17 
11 
The estimated timeline for this study is shown below: ................................................................. 18 
12 
REFERENCES ............................................................................................................................. 19 
13 
Appendix ...................................................................................................................................... 21 
13.1 
Appendix A: Study teams and the participating site for this study ........................................ 21 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1 
LIST OF ABBREVIATIONS 
 
CA  
Cancer Antigen  
CEA  
Carcinoembryonic Antigen 
cfDNA 
cell-free Deoxyribonucleic Acid 
COPD 
Chronic Obstructive Pulmonary Disease  
CRF 
Case Report Form 
CYFRA 
Cytokeratin 19 Fragment Antigen  
DELFI 
DNA Evaluation of Fragments for early Interception 
DNA 
Deoxyribonucleic Acid 
FFPE 
Formalin-Fixed, Paraffin-Embedded 
ICF 
Informed Consent Form 
IRB 
Institutional Review Board 
LDCT 
Low-dose Computed Tomography 
miRNA 
micro–Ribonucleic Acid 
NELSON Dutch–Belgian Lung-Cancer Screening Trial (Nederlands–Leuvens 
Longkanker Screenings Onderzoek)  
NGS 
Next-Generation Sequencing 
NLST 
National Lung Screening Trial  
NSCLC 
Non-Small Cell Lung Cancer 
PI 
Principal Investigator 
PIS  
Participant Information Sheet 
qPCR  
Quantitative Polymerase Chain Reaction 
SCLC 
Small Cell Lung Cancer 
SOP  
Standard Operating Procedure 
USPSTF United States Preventive Services Task Force  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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2 
BACKGROUND INFORMATION AND STUDY RATIONALE 
 
There are two major types of lung cancer, namely non-small cell lung cancer (NSCLC) 
and small cell lung cancer (SCLC), with the former occurring 80% of the time 
(American Cancer Society, 2023). In the year 2020 alone, almost 2.2 million new 
cases of lung cancer were diagnosed worldwide, equivalent to about 6000 cases being 
diagnosed in a day (World Health Organization, 2020). Despite having about the same 
number of diagnoses as breast cancer, lung cancer (1.8 million deaths) caused more 
than twice as many deaths in 2020, when compared to breast cancer (680,000 deaths). 
More alarmingly, the incidence and deaths have been increasing in certain countries 
with lower socio-demographic indexes, as well as in female non-smokers (Ebrahimi et 
al., 2019). Lung cancer is the third most common cancer in Malaysia, but the most 
common cancer in men, accounting for 17% of all cancer diagnosed in men in 2020 
(World Health Organization, 2020). Similar to trends observed globally, the number of 
deaths recorded by lung cancer patients in Malaysia is more than that of breast cancer 
patients, despite having a lesser number of new diagnoses. Further breakdown 
according to the three major ethnic groups in Malaysia, the lifetime risk for lung cancer 
was the highest among Chinese (1 in 51), followed by Malays (1 in 62), and Indians 
(1 in 145) for the years 2012-2016 (Ministry of Health Malaysia, 2019).  
The advancement of technology in health care systems has brought us conveniences 
in early lung cancer screening. Many technologies have been employed for lung 
cancer diagnosis, for instance, sputum cytology, X-ray, and low-dose computed 
tomography (LDCT) imaging (American Cancer Society, 2023). Of all the technologies, 
LDCT has been most frequently used for lung cancer screening. LDCT involves a thin 
radiation beam to create a series of pictures taken from different angles to create 
detailed images of the lungs (American Cancer Society, 2023). In their latest 
guidelines, the United States Preventive Services Task Force (USPSTF) recommends 
annual LDCT screening for adults aged 50 to 80 years old who have a 20 pack-year 
smoking history and currently smoke or have quit within the past 15 years (USPSTF 
et al., 2021). In Malaysia, there is no national lung cancer screening programme. 
However, several public hospitals and many private healthcare institutions provide 
LDCT lung cancer screening services (Ministry of Health Malaysia, 2017). Current 
recommendations for lung cancer screening in Malaysia by the Lung Cancer Network 
Malaysia are for current and former smokers with at least 20 pack years, and non-
smokers who have a family history of lung cancer (Lung Cancer Network Malaysia 
and National Cancer Society Malaysia, 2021). 
Despite its advancements, LDCT does come with its challenges at the patient, provider, 
and medical system levels (Patel et al., 2021). Out of fear of a cancer diagnosis or 
concerns about cost, patients often refuse to undergo an LDCT test (Patel et al., 2021). 
Unlike colon or breast cancer screening, which is based on age, the eligibility for a 
lung cancer screening test is often based on smoking habits. High-risk smoking habits 
are often correlated with lower socioeconomic status among patients. Some obstacles 
faced by these individuals include lesser financial resources, lesser awareness about 
the importance of cancer screening, more difficulties in taking time off from work, etc. 
(Patel et al., 2021). On a medical provider level, some of the challenges include the 
management of detected nodules, quality of screening, and radiologists’ expertise in 

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interpreting the results of an LDCT (Martini et al., 2021). There are also some doubts 
in physicians regarding the effectiveness of LDCT in lung screening, as two large 
randomised trials, the National Lung Screening Trial (NLST) and the Dutch–Belgian 
lung-cancer screening trial (Nederlands–Leuvens Longkanker Screenings Onderzoek 
[NELSON]) reported false-positive rates per screening round of 23.3% and 10.4% 
respectively (National Cancer Institute, 2022). On the other hand, to implement 
widespread lung cancer screening programmes, medical systems must be able to 
cope. This includes having a robust infrastructure, implementing referral policies, 
follow-up recommendations, and so on (Patel et al., 2021). All the points mentioned 
above come with the need for extra resources, which would then create another set of 
challenges in developing and less developed countries, including Malaysia. In 
Malaysia, the introduction of a pilot study to evaluate the feasibility and outcome of 
single LDCT was proven challenging, when it was terminated prematurely due to low 
recruitment from the general public (Rajadurai et al., 2019). This further bolsters the 
argument that it is difficult to promote LDCT lung screening in Malaysia, despite having 
high lung cancer diagnoses and death rates. 
Liquid biopsy is one of the recent medical advancements that can potentially overcome 
the challenges faced by LDCT in the early detection of lung cancer. A liquid biopsy 
involves the isolation of circulating tumour cells, cell-free DNA and RNA, extracellular 
vesicles, etc. from human bodily fluids, like blood, urine, or sputum, collected through 
minimally invasive methods (Lone et al., 2022). Some of the clinical applications of 
liquid biopsy include early cancer detection, disease progress monitoring, detection of 
therapeutic targets, and many more (Lone et al., 2022). In normal conditions, miRNAs 
mainly function in regulating the degradation or translational repression of mRNA 
targets. However, it has been found that miRNA functions have been dysregulated in 
cancer cells, providing a potential to discriminate between miRNAs in a healthy versus 
diseased person (Ying et al., 2020). Recently, our group has developed and validated 
a serum microRNA (miRNA) panel that can be utilized to detect early-stage NSCLC 
(Ying et. al., 2020). Circulating cell-free DNA (cfDNA) are extracellular fragments of 
nucleic acid that are excreted from cancer cells via apoptosis or necrosis (Gao et al., 
2022). Similar to miRNA, cfDNA in cancer patients also exhibit alterations like 
mutations, hyper- or hypo-methylation, chromosomal rearrangements, etc., which can 
be harnessed to detect cancer earlier than traditional screening methods (Gao et al., 
2022). A recent study by Kruusmaa et al. showed that by identifying changes in cfDNA 
methylation, they could detect early-stage lung cancer (Kruusmaa et al., 2021). In 
another study, they showed the ability to detect lung cancer early by evaluating 
fragmentation patterns of cfDNA in the genome, through an approach called DELFI 
(DNA evaluation of fragments for early interception) (Mathios et al., 2021). 
In this study, there will be a focus on cfDNA and miRNA derived from blood. These 
biomarkers and/or signatures are expected to improve current blood-based protein 
markers such as CA15-3, CYFRA 21-1, CA125, and CEA. They may be deployed as 
adjunct tests to enhance the accuracy of imaging diagnostics like X-ray and LDCT. 
 

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3 
STUDY OBJECTIVES AND OUTCOME 
 
3.1 Study Objectives 
 
This study aims to  
a. Identify biomarkers and signatures to differentiate between high-risk and 
lung cancer patients. 
b. Discover blood-based biomarkers that can be used as an adjunct test to 
imaging diagnosis to improve patient follow-up. 
 
3.2 
Study Hypotheses 
 
With existing evidence showing the utility of using liquid biopsy for early cancer 
detection (Klein et al., 2021; Wan et al., 2019; Cohen et al., 2018), we hypothesized 
that cell-free DNA and/or RNA expression changes during disease progression, and 
biomarker signature can be derived from the changes in expression levels. 
 
3.3 
Study Outcome 
 
To discover and validate RNA and methylated cell-free DNA cancer biomarkers that 
can discriminate between high-risk and lung cancer patients. 
4 
STUDY DESIGN 
 
This is an observational study estimated to enrol approximately 400 participants. The 
study is designed to have 2 cohorts A and B, with the number of targeted participants 
as listed in Table 1. Because of possible attrition, the “Target Final Number” column 
illustrates the desired number of participants to have at the end of the study.  
 
Table 1. Number of targeted participants 
Cohort 
Target Enrolment 
Number 
Target Final Number 
A 
200 
160 
B 
200 
160 
 
This study comprises mainly of prospective analysis of blood, with an examination of 
FFPE tissue specimens, if participants undergo biopsy. The estimated start and end 
date of the study is August 2023 through August 2026, with the start date dependent 
on the approval by the Research Ethics Review Committee. Recruitment of 
participants will be carried out in University Malaya Medical Centre (UMMC) in this 
setting (details in Appendix).  
 
In this study, the cohorts are split into two based on whether they are high-risk or highly 
suspicious of having lung cancer (Fig. 1). With consideration to the patient journey 
across the healthcare institutes, there are specific time-points (Fig 1. teal-coloured box) 
that the subject will be enrolled and briefed on the participant informed sheet (PIS) 
which covers the details of the study, objectives, risks, and benefits in participating in 
this clinical study. An informed consent form (ICF) will be signed by the participant if 
he or she agrees to participate in this study. 
 

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Upon completion of the recruitment process, the subject will undergo a blood collection 
at a designated area during Visit 1. A second blood collection will be performed during 
the subject’s next standard of care follow up visit. This will be Visit 2. Blood will be 
processed by trained personnel according to MiRXES’s procedure. This is denoted in 
Figure 1 as the red-coloured box. This will be further elaborated in the study 
methodology below. 
 
 
Figure 1. Recruitment cohorts and their patient workflow. 
 
 
 

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Figure 2. Detailed patient workflow. 
5 
STUDY POPULATION 
 
5.1 Criteria for Recruitment 
 
The description for Cohorts A and B are as follows: 
 
5.1.1 Cohort A 
Subjects in this cohort are recruited if they have either of the following: pre-existing 
chronic obstructive pulmonary disease (COPD) or are found to have lung nodules 
smaller than 11mm, or nodules that are deemed not for biopsy by the attending 
physician. Subjects in Cohort A will undergo a follow-up imaging according to standard 
of care (based on physician’s choice). During follow-up, another blood collection will 
be carried out. If the follow-up X-ray or LDCT shows increase in the size of nodules or 
signs of cancer development, a biopsy may be performed for histopathology diagnosis. 
Subjects may remain in Cohort A, or, if lung cancer is diagnosed, they will be re-
classified to Cohort B.    
 
5.1.2 Cohort B 
Subjects in this cohort are highly suspicious of having lung cancer. From the results 
of the X-ray or LDCT imaging, the subject would have nodules larger than 11mm, or 
show signs of lung cancer. After obtaining consent and collecting blood from the 
subject, he or she will proceed to a CT-guided lung biopsy as per standard of care. 
The biopsied lung tissue will be analysed by histopathology. In an event that lung 
cancer is not diagnosed based on histopathology results, the subjects will be re-
classified to Cohort A. Subjects that were diagnosed with lung cancer will undergo 
another blood collection during their clinical follow up.  
 

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On average in a month, it is expected that 5 to 10 subjects will be recruited for each 
of Cohort A and B. Logistics and administrative matters to identify and enrol subjects 
will be largely driven by the lead PI’s team.  
 
5.2 Inclusion Criteria 
 
5.2.1 Inclusion Criteria for Each Cohort 
 
Table 2. Inclusion criteria specific for each cohort 
Cohort 
Inclusion Criteria 
A 
i. 
Male or female subjects aged 30 and above. 
ii. No previous history of any cancers. 
iii. Able to provide X-ray and/or LDCT results. 
iv. Have one of the following conditions: 
a. COPD 
b. Pulmonary nodules measuring less than 11mm, or deemed not 
for biopsy by attending physician 
v. Willing to go back for a follow-up X-ray or LDCT scan in the next clinical 
follow up as per standard of care. 
vi. Willing to provide up to 30mL of blood in month-0 (first visit) and up to 
21mL in the next standard of care follow up visit (second visit). 
B 
i. 
Male or female subjects aged 30 and above. 
ii. No previous history of any cancers. 
iii. Able to provide X-ray and/or LDCT results. 
iv. Have one of the following conditions: 
a. Suspicious nodules measuring more than 11mm. 
b. Imaging diagnosis suggestive of lung cancer. 
v. Willing to enrol for biopsy for confirmation. 
vi. Willing to provide up to 39mL of blood in month-0 (first visit) and up to 
21mL in the next standard of care follow up visit (second visit). 
 
5.3 Exclusion Criteria 
 
i. Subject has received chemotherapy or radiotherapy for cancer treatment, 
and any other cancer-related treatment. 
ii. Subject is pregnant or lactating (self-declaration). 
iii. Subject is unwilling or unable to provide signed informed consent. 
 
5.4 Subject Withdrawal 
 
Subjects’ participation in this study is absolutely on a voluntary basis. Subjects are 
free to request to be withdrawn from the study at any time, without giving a reason, 
and this will not affect the benefits that they are entitled to. However, the site should 
make a reasonable effort to ascertain the reasons while fully respecting the subject’s 
rights. There will also be instances where subjects are considered unsuitable to 
continue with the study, a decision made together by the research team and the 
Sponsor. These situations might include, but are not limited to, if subjects are 
physically unfit, subjects are unwilling to follow study procedures, subject was unable 
to provide sufficient blood for the research study or if subjects are found to be 
deliberately providing false information that is detrimental to the integrity of the study 
data. 

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5.5 
Blood Sample Requirements 
 
Blood samples meeting the following criteria will be rejected for blood samples 
collected during Visit 1 or Visit 2: 
i. 
Blood samples collected from subjects within an hour after food. 
ii. 
Blood samples collected from subjects who have been administered local or 
general anaesthetics/sedatives, or any agents used for medical imaging (e.g., 
contrast agents) within the last 24 hours before blood draw. 
iii. 
Blood samples collected from subjects who have taken laxatives within the last 
24 hours before blood draw. 
 
Blood samples meeting the following additional criteria will be rejected for only Visit 1 
blood collection: 
i. 
Blood samples collected from subjects who have anti-cancer drug or anti-cancer 
treatment including chemotherapy, surgical removal of tumor, radiation therapy 
and any other cancer-related treatments.  
ii. 
Blood samples collected from subjects who have received blood transfusion 
within the last 6 months before blood draw.  
5.6 
Possible Risks and Discomfort to Subjects 
 
No experimental drugs will be administered in this clinical research study. Blood 
collection by venepuncture, which is a minimally invasive procedure performed in this 
study, carries minimal risk. Possible side effects of blood drawing include faintness, 
inflammation of the vein, pain, bruising, or transient bleeding at the site of 
venepuncture. There is also a slight possibility of infection. A separate third party 
insurance policy will be established to cover research related injury. 
 
The processing of blood samples and molecular analysis will be performed in qualified 
laboratories non-accessible to the recruited subjects. The outcome of the molecular 
analysis will not be reported to the subjects. In addition, the study team and the 
sponsor will not give medical advice to the subjects. Therefore, subjects are not 
exposed to any medical risk. 
 
5.7 
Adverse Events 
 
Adverse events (AEs) are any untoward medical occurrence (e.g. signs, symptoms, 
abnormal laboratory findings, unintended signs) that are temporally associated with 
the human biomedical research. There may or may not be any causal relationship with 
the study procedures performed as part of the research study. 
 
Serious adverse event (SAE) in relation to human biomedical research, means any 
untoward medical occurrence as a result of any human biomedical research which: 
• 
results in death 
• 
is life-threatening 
• 
requires in-patient hospitalization or prolongation of existing hospitalization 
• 
results in or contributes to persistent or significant disability/incapacity 
• 
results in or contributes to a congenital anomaly/birth defect 

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• 
 is an important medical event that may be classified as serious based on 
medical judgement 
 
AEs that are deemed related to the research study will be recorded.  
 
SAEs deemed related (definitely/ probably /possibly) to the research study will be reported to 
the IRB. Related means there is a reasonable possibility that the event may have been 
caused by participation in the research. The investigator is responsible for informing the IRB 
after first knowledge that the case qualifies for reporting. Follow-up information will be actively 
sought and submitted as it becomes available.  
 
5.8 
Possible Benefits 
 
Depending on the groups that subjects are assigned to, recruited subjects may benefit 
from health screening tests, medical tests, or consultations that may inform the 
underlying health risks that the subjects may not be aware of.  
 
5.9 Costs and Payments if Participating in the Study 
 
There will be no cost required from the participants. However, monetary and/or 
material compensation may be given to the participants and supporting healthcare 
professionals.  
 
The research will require clinical information as indicated under Section 6.4 and also 
relevant imaging information for the analysis. This study will provide insights into the 
blood analytes of these cohorts and their concordance with the clinical information.  
 
The investigator will be candid to the participants that the discovery work developed 
from this study may have financial value once approved by regulatory agencies for 
clinical use. However, the participants must be made to understand that the outcome 
of this study will not entitle them to any royalty fees. Further, it will be explained to the 
participants that the acquisition of the blood specimen will not affect their standard of 
care treatment. 
 
6 
STUDY PROCEDURES AND ASSESSMENT 
 
6.1 Enrolment 
 
Any subject will be considered for enrolment provided they meet all the inclusion 
criteria and none of the exclusion criteria. The PIS and ICF will be discussed with the 
subjects. Subjects will be given ample time to consider and ask questions as needed. 
If the subjects are agreeable to the terms of the ICF, it will be signed by the subject, 
the PI or the study team member conducting the informed consent discussion, and the 
witness. The subject will be assigned a subject code to de-identify the subject. 
 
6.2 
Data Collection  
 
The demographics, lifestyle, personal health data, family history of diseases, clinical 
data, response to treatments and other medical records will be collected through 

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medical record review and interviews with participants. The scope of the data to be 
collected will be defined in the case report form completion guidelines for this study 
This de-identified information are then entered using the tablet/laptop available on-site 
with the study team member into the electronic data capture (EDC) system. There will 
be a review and collection of data from participants and the medical record database 
at multiple time points, namely the time of recruitment and subsequently at interim 
monitoring visits. 
 
 
6.3 
Blood Samples Collection 
 
Up to 39mL of blood will be collected at the appointed blood collection point from each 
enrolled subject:  
Visit / Cohort 
Blood Volume 
No. of Blood Tubes 
Visit 1 (Cohort A) 
30 mL 
3 RNA Complete BCT™ tube (9 mL each)  
1 BD Vacutainer® EDTA tube (3 mL) 
Visit 1 (Cohort B) 
39 mL 
4 RNA Complete BCT™ tube (9 mL each)  
1 BD Vacutainer® EDTA tube (3 mL) 
Visit 2 (Cohort A & B) 
21 mL 
2 RNA Complete BCT™ tube (9 mL each) 
1 BD Vacutainer® EDTA tube (3 mL) 
 
The blood collection should be executed in the following blood tube order (Table 4): 
 
Table 4. Sequence of blood collection according to blood tube  
Sequence of Collection 
Type of Blood Tube 
Volume of Blood (ml) 
First 
RNA Complete BCT™ 
9 
Second 
BD Vacutainer® EDTA 
3 
Third 
RNA Complete BCT™ 
9 
Fourth (where applicable) 
RNA Complete BCT™ 
9 
Fifth (where applicable) 
RNA Complete BCT™ 
9 
 
The collected blood will be processed within 24 hours and stored at room temperature. 
Blood tubes will be labelled and processed according to MiRXES’ Standard Operating 
Procedures (SOPs), available in the Appendix section. 
 
Up to two attempts may be executed for the blood drawn and the definition of a 
successful blood drawn is at least 2 x RNA Complete BCT™ and 1 x BD Vacutainer® 
EDTA with no haemolysis. The definition of successful blood drawn requirement 
applies to only Visit 1. If the minimum volume is not collected in Visit 1, the patient will 
be withdrawn and excluded from the study.  
 
6.4 
Clinical Specimens and Key Data Requirements 
 
The table of clinical information to be collected from each cohort is detailed under 
Appendix G. Out of which, critical clinical information listed under the table needs to 
be collected to be considered clinically evaluable. 
 
6.5 
Biomolecules Extraction and Sequencing  
 

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At least two types of biomolecules will be isolated from the samples used in this study. 
 
6.5.1 miRNA 
 
Total RNA from platelet-poor plasma will be isolated using a commercially available 
kit. RNA will be reverse transcribed using multiplex reverse transcription primer pools 
with ID3EAL cDNA synthesis reagents (MiRXES). cDNA will be pre-amplified using 
Augmentation Primer Pools (MiRXES). Singleplex qPCR will then be performed on the 
amplified cDNA samples using a miRNA-specific qPCR assay and ID3EAL miRNA 
qPCR Master Mix (MiRXES).  
 
6.5.2 Cell-free DNA (cfDNA) 
 
cfDNA will be extracted from platelet-poor plasma using commercially available kits. 
The methylation profile of the cfDNA samples will be assessed using hybridization-
based targeted sequencing. 
 
6.5.3 Others 
 
Other biomolecules present in the buffy coat, red blood cells, and/or plasma may be 
isolated and analysed to discover additional biomarkers. 
 
7 
ANALYTICAL METHODS 
 
For miRNA analysis, expression levels of the miRNAs profiled using MiRXES qPCR 
ID3EAL solution will be quantified based on an intra-run standard curve. A set of 
miRNAs with expression levels significantly altered between the comparison groups 
will be identified.  
 
For methylated DNA analysis, the sequencing reads subjected to adapter and quality 
trimming will be mapped to the human genome. The methylation levels of the gDNA 
between the cancer cells and healthy cells will be compared. Similarly, the methylation 
levels of the cfDNA across the healthy control groups and the malignant group will be 
compared, and the differences will be subjected to statistical analysis to identify the 
DNA regions that are differentially methylated at a significant level between the groups.  
 
8 
DATA HANDLING AND RECORD KEEPING 
 
8.1 Clinical Data Collection Forms  
 
A case report form (CRF) is required and must be completed for each subject. The PI 
of the study team will be responsible for the completeness of the form and to review 
and approve all CRFs. CRFs must be signed by the PI or by an authorized staff 
member. These signatures serve to attest that the information contained in the CRF 
is true. At all times, the PI has final personal responsibility for the accuracy and 
authenticity of all clinical and laboratory data entered into the CRF. 
 

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Subject source documents are the physicians’ subject records maintained at the study 
site (CIC UMMC). In this study, the source documents will include the patient hospital 
case notes, physician's charts, and the study source documents. The information 
collected on the CRFs must match these.  
 
8.2 Data Entry and Storage 
 
Any hardcopy documents of this study containing subjects’ information will be stored 
in a designated locked cabinet(s) or room(s) that are accessible only to authorised 
study personnel. The hardcopy data will be transferred into an electronic database for 
record storage. These data will be stored in a secured computer protected with a 
password. The database will not contain subject identifiers. The data linking subject 
identifiers and the subject code will be stored separately.  
 
9 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
 
All data obtained from the study will be kept confidential and be available to the PI, 
research staff, and study coordinators from the study team, including those on the 
delegation log. The study team will maintain the password-protected database linking 
the subject identifiers to unique subject codes, for as long as the samples are stored. 
 
10 ETHICAL CONSIDERATIONS 
 
10.1 Institutional Review Board (IRB) 
 
The PI of each study team will obtain prospective approvals of the study protocol, 
protocol amendments, informed consent forms, and other relevant documents, if 
applicable, from the IRB for the recruitment of participants, collection of samples, and 
data. 
 
Ethical approval for this study will be obtained from the University Malaya Medical 
Centre (UMMC) Medical Research Ethics Committee (MREC), a written informed 
consent will be obtained from volunteer participants prior to the collection of their 
biological samples, demographic data, and other relevant clinical information. 
   
10.2 MiRXES’ IRB 
 
MiRXES will obtain the relevant approval from the assigned Institutional Review Board 
(IRB) for this study. 
 
10.3 Ethical Conduct of the Trial 
 
The study will be performed in accordance with International Conference on 
Harmonization Good Clinical Practice guidelines, the Declaration of Helsinki (2000), 
and applicable local regulatory requirements and laws. All biomedical research 
principles that involve human subjects, such as beneficence, confidentiality, and 
respect for rights and justice, will be construed before the participants in the study. 
 

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10.4 Confidentiality of Data and Patient Records 
 
Study participants will be asked for their written ICF, available in English, and will be 
assured of the confidentiality of their information and test results. The ICF process will 
have to take place on-site. The ICF will state the obligations, compensations, risks, 
and benefits to them as a participant. The clinical specimens collected will be used for 
research purposes. 
 
To maintain the anonymity of the study, information of the participants is maintained 
in adherence to the Malaysian Personal Data Protection Act 2010 (PDPA) of Act 709. 
A unique code will be assigned to each participant to maintain anonymity. This unique 
code will be the only link to the participant's information, which will be stored in a 
password-secured database that can only be accessed by the data manager of the 
research team. It will be the only identifier that can be used by the other members of 
the research team when collecting and analysing their biological samples or when 
requesting information from the data manager. The data manager can share the 
participants’ information with the other members of the research team using coded 
data. An electronic logbook will record who accessed the information and what 
information was obtained. Biological samples will be labelled with the same unique 
code assigned to the participant, and no other identifier information should be included 
in the said label. The biological samples and results of the analysis can neither be 
linked back to the participant nor be used to identify the participants.  
 
To enable evaluations and/or audits from regulatory authorities, the PI will keep 
records, including the identities of all participating subjects (sufficient information to 
link records, e.g., ICFs and hospital records), all original signed informed consent 
forms, copies of all ICFs, source documents, and detailed records of treatment 
disposition. The records should be retained by the PI according to local regulations. 
The PI(s)/Institution(s) will permit study-related monitoring, audits, and/or Institutional 
Review Board review and regulatory inspection(s), for direct access to source 
data/documents. 
 
The blood specimens collected from the study will be processed and shipped to 
Singapore for analysis. The remaining specimen from the analysis may be archived 
and stored for further analysis where required. The storage facility holds the 
responsibility for the clinical specimens and has to ensure that they are properly 
documented and tracked with the integrity of records of the consent and other 
information relating to the participant. A separate institutional review board (IRB) will 
be required, exercised in and by the storage facility. The storage facility will also bear 
the responsibility that the specimens are properly disposed of following recommended 
protocols by the local regulations, if necessary. 
 
The results of this study will be presented to the public through different means, 
including but not limited to radio, newspaper, other periodicals, social media platforms, 
local and international conferences, conventions, and published in scientific or medical 
journals without revealing the participants’ identities. 
 
10.5 Addressing Vulnerability  
 

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To protect the vulnerable population from exploitation, the PIS and ICF are presented 
in English. The investigator will provide supplementary educational measures and 
even interpret and translate the materials for the participants. This is to ensure that the 
research subjects will receive and process the information accurately and express 
considered choices.  
 
Prospective participants who do not give their consent will continue to be treated in 
the usual and customary fashion. Their biological specimens will not be included 
among the samples to be analysed. 
 
Concerns and issues regarding the privacy and confidentiality of participants’ 
information and results of molecular analyses will be addressed appropriately. Benefits 
and risks will be thoroughly and carefully assessed during the course of the clinical 
trial. 
 
11 The estimated timeline for this study is shown below: 
 
Milestone 
Target date 
Research Protocol consensus with all parties 
1-Jun-23 
Equipment and consumables check  
15 Jul-23 
Ethics approval 
31-Jul-23 
Lab personnel training 
15-Aug-23 
Site Initiation Visit – PPUM and Respiratory Clinic 
30-Aug-23 
Study start date 
4-Sep-23 
Send out 10 x Cohort A and 10 x Cohort B to Singapore for verification 
29-Oct-23 
50-subject pulse check 
5-Nov-23 
100-subject pulse check 
4-Feb-24 
200-subject pulse check 
4-Aug-24 
Post recruitment check 
Review on extension of study 
19-Sep-24 
 Target 300 subjects 
2-Feb-25 
Data analysis 
24-Oct-26 
Study wrap up and report 
28-Dec-26 
 
 

 
 
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13 Appendix 
 
13.1 Appendix A: Study teams and the participating site for this study 
 
Lead PI: Associate Prof. Dr. Poh Mau Ern 
Department of Medicine 
Faculty of Medicine 
University of Malaya 
 
Co-PI: 
Dr Diana Ong Bee Lan 
Department of Pathology 
Faculty of Medicine 
University of Malaya 
 
Sub-Investigators: 
Associate Prof. Dr Pang Yong Kek 
Dr Wong Chee Kuan 
Dr Tan Jiunn Liang 
Dr Soo Chun Ian 
Dr Loh Thian Chee 
Dr Chin Ka Kiat 
Dr Vijayan Munusamy 
Dr Sia Leng Cheng 
Dr Nur Husna Ibrahim 
 
 
 
Study site: 
 
University Malaya Medical Centre (UMMC) 
Lembah Pantai, 59100 
Kuala Lumpur, Malaysia.