Hepatocellular Carcinoma (HCC) is the fifth most common cancer world-wide. It is particularly prevalent in Asia, and its occurrence is highest in areas where hepatitis B is prevalent, indicating a possible causal relationship. Follow up of high-risk populations such as chronic hepatitis patients and early diagnosis of transitions from chronic hepatitis to HCC would improve cure rates. In most cases HCC is detected late resulting in increased mortality and morbidity.

The purpose of this study is to develop and test non-invasive biomarkers based on methylation changes in PBMC and circulated tumor DNA in hepatocellular carcinomas patients.

Inclusion Criteria:

• Confirmed diagnosis of HCC by EASL-EORTC guidelines
• Confirmed hepatitis B diagnosis for HepB patients using AASLD practice guidelines.

Exclusion Criteria for HCC:

• Cirrhosis, any other known inflammatory disease (bacterial or viral infection with the exception of hepatitis B or C, diabetes, asthma, autoimmune disease, active thyroid disease) which could alter T cells and monocytes characteristics
• Other cancers.

Exclusion Criteria for HepB:

• Diagnosis of HCC or any other cancer.

Exclusion Criteria for Healthy Controls:

• Any known inflammatory or infectious disease including HepB and HepC
• Chronic diseases,
• Cancer
• Medications or drug use

HCC staging will be diagnosed according to EASL-EORTC Clinical Practice Guidelines: Management of hepatocellular carcinoma. The patients will be divided into four groups, including Stage 0 (1) (n=100), stage A (2) (n=100), stage B (3) (n=100) and stage C+D (4) (n=100). As controls we will recruit chronic hepatitis B (n=100), which diagnose will be confirmed using AASLD practice guideline for chronic Hepatitis B, and healthy sex and age matched controls (n=100).

402 participants were recruited from the Dhaka area to the study included 49 healthy controls, 51 Chronic hepatitis B patients and 302 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=76)

Our observational study, focused on identifying biomarkers for HCC detection, did not involve the collection or monitoring of adverse event data. The duration for participant involvement spanned from the study start date of August 20, 2018, to the primary completion date of June 1, 2020. During this period, spanning approximately 1 year and 9 months, there were 0 participants at risk for adverse events due to the non-interventional nature of the study.

All-Cause Mortality, Serious, and Other (Not Including Serious) Adverse Events were not monitored or assessed because the study was non-interventional and focused solely on testing liquid biopsy methylation markers. Therefore, no participants were at risk for adverse events.

Inherent challenges of our study included a regional focus and a reliance on observational data, which may limit broader applicability and prevent establishing causal relationships. Technical aspects, like the exclusion of participants due to insufficient sequencing depth (less than 100 reads per gene for five out of 402 participants), reflect constraints in data robustness. These elements represent areas for potential enhancement in future research