Clinical Trials on Detection of Hepatocellular Carcinoma With Non-invasive Method Based on DNA Methylation of Circulated Tumor DNA, PBMC and T Cells
Conditions: Hepatocellular Carcinoma
Sponsor: HKGepitherapeutics

Title: HCC Screening using DNA methylation Changes in ctDNA  
 
ClinicalTrials.gov Identifier: NCT03483922 
Date: January 11, 2022 
 
Clinical study design 
 
Study protocol was approved by IRB board of icddr,b (Dhaka, Bangladesh) study protocol PR- 
18025. 402 participants were recruited from the Dhaka area to the study included 49 healthy 
controls, 51 Chronic hepatitis B patients and 303 HCC patients from stages 0 to D (HCC O n=2, 
HCCA n=32, HCC B n=86, HCC C n=106, HCC D n=77 (See Table 2 for demographics). The 
age of the healthy control and the chronic Hepatitis B (CHB) groups were significantly younger 
27 than the HCC group, however there was no significant effect of age on HCC prediction by 
DNA 
methylation as determined by a logistic regression in either the HCC or control groups. However, 
in the CHB group age had a significant effect on HCC prediction by the DNA methylation test, 
consistent with the idea of a higher probability for older patients with chronic hepatitis to convert 
to HCC. There were no differences between the groups in the sex distribution. However, there 
was a somewhat significant lower alcohol use and higher fraction of smokers in the HCC groups 
(Table 2). HCC staging was diagnosed according to EASL–EORTC Clinical Practice 
Guidelines: Management of hepatocellular carcinoma. Hepatitis B diagnosis was confirmed 
using AASLD practice guideline for chronic Hepatitis B 
(https://www.aasld.org/publications/practice-guidelines). All participants were properly informed 
about the study and have signed the informed consent form approved by the icddr,b IRB. 
Inclusions criteria were participants of either sex 18 to 70 years of age, confirmed diagnosis of 
HCC using EASL-EORTC guidelines and chronic hepatitis B using AASLD guidelines, 
nonmetastatic 
liver cancer, Hepatitis B surface antigen positive by ELISA and persistence of > 6 
months. Exclusion criteria were unwilling or unable to provide informed consent, unwilling or 
unable to comply with requirements of protocol, participation in a different clinical trial 
investigating a vaccine, drug, medical device or medicinal procedure less than 4 weeks preceding 
the current study, planned participation in another clinical trial during present study period, 
known case of cirrhosis, any other known inflammatory disease (bacterial or viral infection with 
the exception of hepatitis B or C), known case of diabetes, asthma, autoimmune disease, any 
other diagnosed cancer, for healthy controls any known inflammatory or infectious disease 
including Hepatitis B and Hepatitis C and any diagnosis of chronic disease, cancer medication 
use or drugs of abuse. Patients were assigned an ID that was kept confidential according to 
 
hospital regulations and identity was revealed only to approved hospital personnel. Participants 
provided consent for DNA methylation biomarker research. Blood sample collection ad plasma 
separation was performed at icddr,b in Dhaka Bangladesh and was then shipped to HKG 
epitherapeutics for further analysis. The HKG epitherapeutics lab team was blinded on the 
identity of the samples throughout the lab analytic procedures. Data was then analyzed in 
Montreal and shared with icddr,b who provided the results to the respective clinical personnel. 
 
Preparation of CF plasma DNA 

 
Blood was collected in 9-ml tubes containing K3-EDTA and processed within 1 h. Plasma and 
peripheral blood monocyte separation was performed according to GE Healthcare Cat No 
71=7167-00 protocol. Plasma was frozen and shipped. Plasma samples (1 ml) were thawed, and 
DNA was extracted by previously described guanidine isothiocyanate method[43] and binding to 
silica magnetic beads followed by 80% ethanol washes and water elution. 
 
Multiplexed Targeted DNA methylation Illumina amplicon sequencing 
 
Bisulfate conversion was performed using EZ-96 DNA Methylation MagPrep (D5041, Zymo 
Research) followed by two rounds of polymerase chain reaction. For the first round we used 
primers that included an anchoring sequence and sequences targeting cg02012576 (CHFR), 
cg03768777 (VASH2), cg05739190 (CCNJ), cg24804544 (GRID2IP) and cg14126493 (F12) 
using Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad Laboratories, CA, USA) (the primers are 
available upon request). 5 microliters of the first PCR reaction were subjected to a second round 
of PCR amplification using primers containing indexes for barcoding the samples (the primers 
are available upon request). PCR products were pooled, and the pooled library was then purified 
twice using AMPure XP Beads (Beckman Coulter Life Sciences, CA, USA) and quantified by 
RealTime PCR using NEBNext® Library Quant Kit for Illumina (New England Biolabs, MA, 
USA). 
Sequencing was performed on the Illumina platform using MiSeq Reagent Nano Kit V2 
(Illumina, CA, USA). 
 
 
Statistical Analysis Plan 
 
Significant difference from the control group was tested by Kruskal-Wallis non-parametric one-
way ANOVA and was adjusted by Dunn’s multiple comparisons test. The two HCC stage O 
samples were included for statistics in the HCC A group. ROC was computed using the ROC test 
in GraphPad.